Table of contents
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TABLE OF CONTENTS . 1
WORKING WITH RECOMBINANT-DNA TECHNIQUES UNDER GMT AND VMT CONDITIONS 7
GMT (Good Microbiological Techniques) 7
VMT (VEILIGE = SAFE MICROBIOLOGICAL TECHNIQUES). 8
VMT workrules and procedures. 8
TECHNICAL MANUAL I EQUIPMENT AND MEDIA 10
I.1 INTRODUCTION 11
I.2 WHERE CAN I FIND CHEMICALS, KITS, ENZYMES, WASTE CONTAINERS, …. 11
I.3 BUFFERS AND COMMON SOLUTIONS 11
I.4 MEDIA FOR BACILLUS SUBTILIS 14
I.5. MEDIA FOR LACTOCOCCUS LACTIS 17
TECHNICAL MANUAL II COMMON BASIC METHODS 19
II.1 BACTERIAL- AND BACTERIOPHAGE STRAINS 20
1.1 Starting material, pure culture 20
1.2 Storage of strains 20
II.2 WORKING UNDER STERILE CONDITIONS 21
II.3 STERILISATION; MELTING AGAR(OSE) 21
3.1 Pression cooker 21
3.2 Magnetron 22
II.4 STERILISATION WITH THE HELP OF MEMBRANE FILTERS 22
II.5 PIPETTING 23
5.1 Glass pipettes (for standard dilution’s, plating etc.) 23
5.2 Micropipettes 23
II.6 DILUTION SERIES 24
II.7 POURING AND DRYING AGAR PLATES 24
II.8 PLATING OUT BACTERIA AND BACTERIOPHAGES 25
8.1 B. subtilis and E. coli 25
8.2 Protoplasts 25
8.3 Transferring colonies to test plates by tooth picking 25
8.4 Bacteriophages (of B. subtilis) 26
II.9 CENTRIFUGING AND WASHING BACTERIAL CELLS 27
II.10 DIALYSING 27
II.11 WASHING OF PHENOL 28
Wash procedure 28
TECHNICAL MANUAL III TRANSFORMATIONS AND TRANSFECTIONS 29
III.1 TRANSFORMATION OF COMPETENT B. SUBTILIS 30
1.2 Competent culture 30
1.3 Transformations 30
1.4 Transfections 31
1.5 Transformation protocol A from F.Kunst 31
III.2 PROTOPLAST TRANSFORMATION (B. SUBTILIS) 32
2.1 Protoplast Transformation in general 32
2.3 DM3 agar 33
2.4 Protoplast-generatiom 33
2.5 Transformation 34
III.3 TRANSFORMATION OF E. COLI WITH PLASMID DNA 34
3.1 Strains 34
3.2 Competent culture CaCl2 method 34
3.3 Transformation 35
III.4. ELECTRO TRANSFORMATION OF E. COLI 35
4.1 Preparation of cells 35
4.2 Protocol for Electro-transformation using the BioRad Gene Pulser 36 III.5. ELECTRO TRANSFORMATION OF LACTOCOCCUS LACTIS 36
5.1 Preparation of cells 36
5.2 Electroporation 36
5.3 Buffers 36
TECHNICAL MANUAL IV DNA ISOLATIONS 37
IV .1 CHROMOSOMAL DNA 38
1.1 Chromosomal DNA for transformations, not for enzyme reactions 38
1.2 Chromosomal DNA Lactococcus lactis 39
1.3 Chromosomal DNA (for enzyme reactions) 39
IV.2 BACTERIOPHAGE DNA 40
2.1. The preparation of a sterile bacteriphage stock 40
2.2 Large lysates (used for DNA isolations) 41
2.3 Continued purification of lysate’s 41
2.4 Purification and concentration of bacteriophage using CsCl density gradient centrifugation. 42
2.5 Isolating the bacteriophage DNA 42
2.6 Isoletion of bacteriophage M13 DNA template 43
IV.3 "MINIPREP" PROCEDURE FOR SMALL SCALE PLASMID ISOLATION 43
3.1 Boiling method 43
3.2 Alkaline (Birnboim) "miniprep" for B.subtilis 44
3.3 Alkaline (Birnboim) miniprep for E. coli 46
3.4 Alkaline (Birnboim) "miniprep" for L. lactis 46
3.5 Fast, one-step miniprep procedure for E. coli 46
IV.4 BIG SCALE PLASMID ISOLATIONS (B.SUBTILIS / E. COLI) 47
4.1 "Maxi prep" method 47
4.2 Alternative method (B.subtilis) 48
4.3 PEG precipitation off plasmid DNA from an CsCl solution 49
IV.5 BIG SCALE PLASMID ISOLATION L.LACTIS 49
IV.6 CALCULATING DNA CONCENTRATIONS 50
6.1 the Dische reactie 50
6.2 Absorption at 260 nm with the Spectrophotometer 51
6.3 Estimating using gel-electrophoresis 51
6.4 Estimating by means of ethidiumbromide fluorescence 51
TECHNICAL MANUAL V GELELECTROPHORESIS 53
V.1 POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) 54
1.1 10% AA gel 100 ml 54
1.2 Pouring the gel 54
1.3 Electrophoresis 54
1.4 Staining and photography of the gel 55
V.2 AGAROSE GEL ELECTROPHORESIS 55
2.1 Separation of linear DNA fragments and plasmids 55
2.2 Buffer 55
2.3 Electrophoresis trays 55
2.5 Pouring the gel 56
2.6 Sample preparation and electrophoresis 56
2.7 Reference markers 57
2.8 Photography using a Polaroid camera 57
2.9 Photography using a geldocumentation system (Video Image Pictures camera) 57
V.3 DNA FRAGMENT ISOLATION FROM AGAROSE GELS 58
3.1 High Pure PCR Product Purification Kit (Roche) 58
3.2 Freeze-squeeze method 59
3.3 Band Intercept Method Using S&S NA 45 DEAE Membrane 60 3.4 Electro-elution with the help of open dialysis membrane 61
3.5 Electro-elution in a closed dialysis membrane tube 61
3.6 Purification with "Prep-A-GeneTm" kit 62
TECHNICAL MANUAL VI RECOMBINANT DNA TECHNIQUES 65
VI.1 RESTRICTION-ENZYMES, RESTRICTIONS 66
1.1 General remarks 66
1.2 Unit definition; complete and partial digests 66
1.3 Inactivation of restriction enzymes 67
1.4 Restriction buffers 67 1.5 Restriction sites 68 1.6 Star Activity 68 VI.2. LIGATION 69 2.1 Description 69 2.2 Reactionbuffer 69
2.3 The ligation reaction 69
VI.4. MAKING 3'-PROTRUDING ENDS BLUNT WITH T4 DNA POLYMERASE 71 VI.5. DEPHOSPHORYLATION REACTION WITH ALKALINE PHOSPHATASE 72 VI.6. EXTRACTION OF DNA WITH PHENOL OR PHENOL/CHLOROFORM 72
VI.7. DNA PRECIPITATION ON MICROSCALE WITH ETHANOL 74
VI.8. PEG PRECIPITATION FOR SELECTIVE REMOVAL OF SMALL DNA FRAGMENTS 75
TECHNICAL MANUAL VII SPECIAL RECOMBINANT DNA TECHNIQUES 76
VII.1 DNA BLOTTING (SOUTHERN HYBRIDISATION) 77
1.1 Description 77
1.2 Protocol 77
VII.2 PREPARATION OF TOTAL LYSATES AND THE DETECTION OF SSDNA IN ROLLING-CIRCLE PLASMIDS 80
2.1 Preparation of total lysates 81
2.2 Hybridisation and detection of ss DNA via ECL 82
VII.3. SITE-DIRECTED MUTAGENESIS (SDM) 83
3.1 Procedure 83
3.2 Protocol of the SDM procedure 85
3.3 Synthesis and purification of mutagene oligonucleotides 87
3.4 Software 87
VII.4. POLYMERASE CHAIN REACTION (PCR) 87
4.1. Screening transformants from the plate: colony PCR 88
4.2. Site-directed mutagenesis 90
4.3. Random mutagenesis with MnCl2 90
VII.5. GELRETARDATION (GELSHIFT) ASSAY. 91
5.1 Promoter labelling 91
5.2 Binding 91
5.3 Gel electrophoresis 91
VII.6. PREPARATION OF CELL-FREE EXTRACTS L.LACTIS 92
VII.7. PLATE CONJUGATION OF LACTOCOCCI 92
VIII.1 RADIOACTIVE SEQUENCING 94 1.1 introduction 94 1.2 Sequencing reactions 94 1.3 Sequencing tips 95 1.4 The Gel 95 1.5 Analysis 97
VIII.2 NON RADIOACTIVE SEQUENCING 98
2.1 Introduction: 98
2.2 Primers 98
2.3 DNA 98
VIII.3 THERMO SEQUENASE FLUORESCENT LABELLED PRIMER CYCLE SEQUENCING KIT WITH 7 -DEAZA-DGTP 99
3.1 Background And References 99
3.2 Components Of The System 100
3.3 Cycle Sequencing 101
3.4 Additional Information 103
VIII.4 REPROGELTM FOR POLYACRYLAMIDE GEL ELECTROPHORESIS WITH THE ALF® FAMILY OF INSTRUMENTS103
4.1. Introduction 104
4.2. Sample preparation 104
4.3. Cleaning the cassette 104
4.4. Placing the cassette in ReproSet TM 104
4.5. Preparing the gel solution 104
4.6. Casting the gel 105
4.7. Polymerising ReproGel 105
4.8. Running parameters 105
VIII.5 ALFEXPRESS®II SHORT INSTRUCTION 107
5.1 Clean the gel cassette components 107
5.2 Apply Bind-Silane 107
5.3 Assemble the gel cassette 107
5.5 Cast the gel 107
5.6 Place the gel cassette into the instrument 108
5.7 Load the samples and starting the run 108
5.8 Start the run 108
5.9 End the run 108
TECHNICAL MANUAL IX PROTEIN TECHNIQUES 109
IX.1 PROTEIN ELECTROPHORESIS AND DETECTION METHODS 110
1.1 Introduction 110
1.2 Preparation of a PAA-protein gel 110
1.3 1-D Protein gels 111
1.4 Coomassie staining 111
1.5 Silver staining of proteingels 111
IX.2 WESTERN BLOT 112
2.1 the blotprocedure 112
2.2 Alternative procedure with "pvdf immobilon" membrane 113
2.3 Semi dry blotting 113
2.4 Alkaline phosphatase staining 114
2.5 Western-Light 115
IX.3 DETERMINATION OF PROTEIN CONCENTRATIONS 115
3.1 Bradford protein detection 115
3.2 Lowry protein detection 116
IX.4 ENZYME ASSAY 116
4.1 α-Amylase assay with starch azure 116
4.2 β-Lactamase assay 117
4.3 β-Galactosidase assay 117
4.4 Isocitrate dehydrogenase assay(bacillus) 118
4.5 Protease assay with azocoll 118
5.1. Introduction : 120
5.2 Vectors : 120
5.3 Procedure : 120
5.4 pTrcHis 122
5.5 pET32 124
IX.6.GREEN FLUORESCENT PROTEIN (GFP). 126
6.1 Introduction : 126
6.2 Vectors : 126
IX.8 DATABASE SEARCH 128
TECHNICAL MANUAL X RNA TECHNIQUES 130
X.0 GENERAL CONDITIONS 131
Purification and analysis of Messenger RNA from prokaryotic Cells 131
X.1 TOTAL RNA ISOLATION (MACALOID METHOD) 131
1.1 Procedure: 131
1.2 macaloid 2% : 131
X.2 RNEASY PROTOCOL FOR THE ISOLATION OF TOTAL RNA 132
X.3 RNA ELECTROPHORESIS 132
X.4 NORTHERN BLOTTING 134
For RNA s longer then 2.5 kb: 134
X.5 DIG LABELING 134
Use DNA from 200-2000 bp as a probe 134
DIG detection Procedure: 134
Reagens and solutions: 135
X.6 RADIOACTIVE LABELING NORTHERN BLOTS 135
Labeling reaction 135 Standard assay 136 hybridization buffer 136 20x SSPE 136 100x Denhardt’s 136 Nonhomologous DNA 136
X.7 1ST STRAND CDNA SYNTHESIS KIT FOR RT-PCR (AMV) 138
Master mix : 138 Reaction: 138 X.8 RT-PCR 138 Procedure: 138 Example PCR: 138 X.9 PRIMER EXTENSION: 138 Procedure 138
Alternative primer extension with radioactive end-labeling of the primer. 139
TECHNICAL MANUAL XI KITS 140
XI.1 PLASMID MIDI PROTOCOL 141
Protocol 141
XI.2 QIAQUICK PCR PURIFICATION KIT PROTOCOL FOR PURIFICATION OF PCR PRODUCTS 141
Protocol 141
XI.3 DNA EXTRACTION FROM AGAROSE GELS 142
Procedure 142
TECHNICAL MANUAL APPENDIX 144
Alphabetical List of Restriction Enzymes 145
Isoschizomers 148
Restriction Enzymes Suitable for Pulsed-Field Gel Electrophoresis (PFGE) 151
Heat Inactivation of Restriction Enzymes 154
Compatible Cohesive Ends 154
Description of DNA Methylation 155
SuRE/Cut Buffer System for Restriction Enzymes 156
Restriction Enzyme Activity in SuRE/Cut Buffer System 157
Star Activity 159
Nucleatide codes 159
Working with recombinant-DNA techniques under GMT and VMT conditions
GMT (Good Microbiological Techniques)
GMT conditions apply to all homologue cloning experiments in Bacillus subtilis, Lactococcus lactis and
Escherichia coli. Also cloning of Bacillus DNA in E. coli (and all other combinations of subtilis, lactis
and coli ) and the use of bacteriophages and plasmids using these bacteria as a host apply to GMT conditions. A special room for cloning experiments that require more stringent demands is in the cellar of wing A. In the normal laboratory rooms experiments on GMT-level are allowed to carry out when complying with the following rules:
1 Ware a labcoat. Comply with the rules for standard microbiological work.
2 Work carefully and properly; prevent contamination.
3 Clean and sterilise materials used for GMT-work before returning them to the central stock or kitchen.
4 Decontaminate your benchtop regularly with 70% alcohol (always at the end of the day).
5 Throw garbage (petri dishes, ependorf tubes, tips, etc.) in autoclavable plastic bags, and autoclave them before they are carried away. 6 Separate the GMT-materials from uncontaminated materials; thus
preventing unnecessary accumulation of dirty glassware or garbage. 7 Use micropipettes: do not pipette with your mouth.
8 See to it that only the tips are contaminated and not the pipettes. If so clean them with 70% alcohol.
9 Use glaspipettes only with pipetting balloon.
10 Place the glaspipettes after use in buckets with decontaminating solution (water + chlorextabletts).
11 Eating, drinking, smoking and the storage of food in labs for GMT-work is not allowed.
12 Wash your hands regularly, if necessary with 70% alcohol. 13 In doubt: ask the assistants.
VMT (Veilige = safe Microbiological Techniques).
VMT workrules and procedures.
1 Doors and windows should be closed during the activities on the lab.
2 The workplace should be clean and proper. The workplace should be exposed to chemical or biological agents as little as possible.
3 The surface of the worktables should be decontaminated at the end of each work day, at the end of each experiment and if there is spilling.
4 After spilling all parts of the contaminated space should be decontaminated. 5 All accidents and unwanted events should be reported in a logbook.
6 Only right certified people are allowed to carry out the duties. The staff should get the appropriate technical and vocational training.
7 A labcoat or other working clothes should be worn. It is not allowed to wear these cloths outside the working room.
8 During the work it is not allowed to wear rings, bracelets, wristwatch's etc. Personal equipment should be stored outside the room.
9 Eating, drinking, smoking and the storage of food in labs for VMT-work is not allowed.
10 Direct contact between hands and the face should be avoided. The use of paper tissues is recommended.
11 The hands should be washed after working with GMO's and in addition before leaving the working room.
12 Use micropipettes: do not pipette with your mouth.
13 The production of aerosols should be avoided in every situation (centrifugation and mixing in closed tubes, first dry wet öses before glowingout). The use of injection needles and syringes is only allowed when there is no alternative methode. 14 Glass ware and instruments that have been in touch with GMO's heve to be
sterilised or decontaminated before washing, re-use or discarding; before carrying away all biological garbage should be sterilised or decontaminated.
15 If contaminated materials are sterilised or decontaminated outside the working room they should be shipped in unbreakable, closed containers.
Technical Manual I
Equipment and media
I.1 INTRODUCTION 10
I.2 WHERE CAN I FIND CHEMICALS, KITS, ENZYMES, WASTE CONTAINERS, …. 10
I.3 BUFFERS AND COMMON SOLUTIONS 10
I.4 MEDIA FOR BACILLUS SUBTILIS 13
I.1 Introduction
The Department of Molecular Genetics has a lot of equipment which is placed in the lab rooms, in the sequence lab, in the arraylab, in the coldrooms, and in the hotrooms. . (Wing A, ground floor and basement)Some of the most common equipment is present in the practical room and can be used by the students. The other equipment is reserved for the personnel of MolGen but can be used if it is not in use, ask the practicum assistants to help you. Try not to interfere with the work of others on the department. Take notice of the following points:
o Let the assistants instruct you on the operation of the apparatus. o Sign in on a apparatus you want to use to get priority in the usage. o Clean up the area and the apparatus after use.
I.2 Where can I find chemicals, kits, enzymes, waste containers, ….
All Chemicals can be found in room A 0043. The enzymes are in the freezer on the corridor. To order new enzymes or chemicals contact Arie Hamminga in room A 0043. Kits are in the corridor cupboard next to room A0031. Chemical waste can be put in the suitable waste containers in the labrooms or in the big container (solid waste) in room A0039. Bacterial waste should be put in autoclavable sacks in each lab and sterilised before discarding.
I.3 Buffers and common solutions
Composition - preparation Antibiotics Filter sterilise all solutions
Ampicilline 2.0 mg/ml ( 40 x in water) 5.0 mg/ml (100 x only for E.coli)
50 mg/ml (1000 x; E.coli; add a drip of NaOH to dissolve) Kanamycine 0.5 mg/ml (100 x for E.coli and B.subtilis)
Chloramphenicol 0.5 mg/ml (100 x for E.coli and B.subtilis).
2.0 mg/ml (2000x for L.lactis) First make a solution of 34 mg/ml Ethanol and dilute this further in water.
Erythromycine 0.2 mg/ml (100 x for B.subtilis)
10 mg/ml (100 x for E. coli). First make a solution of 20 mg/ml Ethanol and dilute this further in water,
store at -20°C
Tetracycline 1.5 mg/ml (100 x for E.coli and B.subtilis) Spectinomycine 10 mg/ml in water
Some antibiotics are not stable. Divide the stock in small portions and store them in the freezer (-20°C).Use the thawn portion only for a few weeks. Don't use agarplates with antibiotics that are older than 1 week.
ATP 20 mM
Sterilise with membrane filter and store at -20°C in 100 µl portions.
E.coli
10 mM EDTA
25 mM Tris-HCl (pH 8.0)
Add just before use: lysozyme 2 mg/ml Birnboim A B.subtilis 20% sucrose 10 mM Tris-HCl, pH 8.1 10 mM EDTA 50 mM NaCl store at 4°C
Add just before use: lysozyme 2 mg/ml. Mix well.
Birnboim B 0.2 N NaOH + 1% SDS
Prepare this solution from 10NNaOH and 25% SDS. The solution is stored at Room Temperature.
Birnboim C 60.0 ml 5 M K-acetate 11.5 ml Acetic Acid 28.5 ml H2O
pH 4.8; store at 4°C.
CaCl2/MgCl2 mix Used for hunger medium (I.D). Stock: 50mM CaCl2; 250mM MgCl2 (100x) Chloroform:isoamylalcohol 24:1
Dithiothreitol (DTT)
0.1 M
Freeze in portions of100 µl. Use a fresh portion each time.
DNA-ase I Stock 2 mg/ml in 1 M MgCl2 (100 x). Freeze in small portions (-20°C). EDTA 0.5 M in H2O (pH ± 8.0), neutralise with concentrated. NaOH-solution.
Ethanol 70% in Demi
96%
Ethidiumbromide Stock 5 mg/ml: This is 50.000 x concentrated stock solution for staining DNA-gels. Use for standard DNA agarose gels 1 µl per 50 ml agarose sol.
Fe-ammonium-citrate
Stock: 0.11gr/100 ml (1000x). prepare fresh every week For minimal growth medium (I.D.).
glycerol sterile 87% Sterilise 15 min, 15 lbs. Use this for making freezer stocks of Bacterial cultures 10% glycerol.
IPTG 100mM in Dest
K-glutamate stock 1 M (200x). For minimal growth medium (I.D.); Ligation buffer Supplied by Roche
Loading buffer (for agarose gels)
Mixture for gelelectrophoreses (reference color solution). Stock 10x contains:
50 % glycerol
0.05 % brome phenolblue 100 mM EDTA
MgCl2 1.0 M
Na-acetate 3.0 M pH 5.2 Adjust pH with concentrated Acetic Acid
NaCl 5.0 M
NaOH 10 N
Phenol-washed See IV.11
Pronase Stock 10 mg/ml, 33x concentrated. For the isolation of plasmid DNA. Incubate 1 hour at 37°C and store in small portions in the freezer (-20°C).
Proteinase K 20 mg/ml in 10 mM Tris-HCl (pH 8.0), EDTA 1 mM (=100 x). Store in small portions in the freezer (-20°C), 100 µl.
Restriction buffers see VIII.2.d
RNAase I Stock 10 mg/ml T10E1 buffer (100 x). Heat 10 min at 90°C after preparation to inactivate any DNAase activity. ). Store in small portions in the freezer (-20°C). RNAase-mix Same as RNAase I but add after heating 2500 U/ml RNAase T1. (100 x)
SDS Sodium dodecyl sulfate: 20% (w/v)
STE Used for cleaning DNA with the 'spun column'. 10 mM Tris, pH 8.0
1 mM EDTA 100 mM NaCl TAE-buffer Buffer for agarose gels.
To prepare 1L 50 X buffer: 249 gr Tris-base 57.1 ml acetic Acid
100 ml EDTA (0.5 M, pH 8.) add water to 1000 ml
TBE-buffer Most common electrophoreses buffer for agarose gels. Usually made 10 x concentrated (for PAGE this buffer is 20 x concentrated). 5x stock is available in the kitchen. To prepare 1L 50 X buffer: 108 g Tris 55 g boorzuur 8.3 g EDTA pH 8.3 add water to 1000 ml TBT buffer For resuspending bacteriophage's.
100 mM NaCl 100 mM Tris-HCl 10 mM MgCl2
TE-buffer For extractions and dissolving DNA. 10 mM Tris-HCl, pH 7.4 1 mM EDTA
Aanmaken als 10x geconc. forraad (100 mM Tris-HCl; 10 mM EDTA) TES-buffer For isolation of plasmid DNA
10 mM Tris-HCl, pH 7.4 10 mM EDTA
200 mM NaCl Tris-HCl 1.0 M, pH 7.4 and pH 8.0.
Weigh the TRIS and dissolve it in a little less than the desired volume. Adjust the pH with concentrated HCl. Adjust the volume with water.
XGal 2% of 4% in DMF
I.4 Media for Bacillus subtilis
Some media are prepared in the kitchen. Ensure yourself in time if there is enough for your experiment. If necessary prepare the media yourself.
media preparation
Casaminoacids 2% solution for growth media;
5% solution for Protoplast transformations (V.2b) Growth factors End concentration in media
Amino acids 20 µg/ml Nucleotides 20 µg/ml vitamins 0.5µg/ml Stock solutions
Make if possible a 100 x concentrated (2 mg/ml for amino acids and nucleotides and 0.05 mg/ml for vitamins riboflavine and nicotinicacid) Exception : Tyrosine dissolves bad, make a 50 x concentrated stock (1 mg/ml).
Sterilisation
30 min at 100°C (steaming).
Remark : A common used strain is Bacillus 8G5 which needs 8 growth factors: tryptophane, tyrosine, adenine, histidine, nicotinic acid, uracil, riboflavine and methionine. Preferably make a 50x stock solution because tyrosine is one the growth factors.
GSC
SSC DSC
Concentrated Saline Citrate: 1.50 M NaCl +
0.15 M tri sodium citrate Standard Saline Citrate: 10 x diluted GSC Diluted Saline Citrate: 100 x diluted GSC
SSC is used for the lysis of B.subtilis cells in DNA isolations and in the dissolving and dilution of chromosomal DNA for transformations. SSC is also used with Southern hybridisations (IX.1).
Hunger medium Used for diluting (competent) B.subtilis cultures. Also suitable for the dilution of bacteriophage suspensions.
100 ml Spizizens salts (SZ) 2.5 ml glucose (Analar, 20%) Minimal agar 2% in aqua dest (plating cells)
Minimal agar medium
Is used as a selective medium in transformations with chromosomal DNA of B.subtilis. 5 to 6 plates are poured from 100 ml.
Per 100 ml:
75 ml molten minimal agar (2% solution) 20 ml 5x SZ
5 ml glucose solution (20%)
1 ml 100x Stock solution of all wanted growth factor's; or 2 ml 50x Stock solution of all wanted growth factor's possible:
1 ml PEP-AZ
0.1 ml Fe-ammonium citrate(1000x I.C.) Minimal growth
medium
100 ml Spizizen's minimal Salts (SZ; I.D.) 2.5 ml Analar glucose (stock solution 20%)
1 ml casein hydrolysate solution (stock solution 2g/100 ml)
Growth factors (for auxotrofe mutants): 1 ml of 100x Stock solution (or 2 ml of a 50x stcock solution.)
PAB 4 x
Penassay broth = antibiotic medium 3
For B.subtilis protoplast transformations van (V.2) 7 g Bacto Penassay Broth in 100 ml aqua dest sterilise: 15 min, 5 lbs
PEP-AZ Mixture of non-essential amino acids. Often added to minimal agar to speed up the growth of colonies, for example after transformation with chromosomal DNA. Composition
100x concentrated stock-solution containing 1 mg/ml of each of the following amino acids: glycine, asparagine, valine, glutaminezuur, leucine, asparaginezuur, isoleucine, proline, phenylalanine, serine, alanine, threonine, glutamine.
When you select on transformants for auxotrophe markers the corresponding aminoacid should not be in the AA-mixture of course. This mixture is suitable for almost all of the strains used in our lab.
Phosphate solution
Used for protoplast transformations (V.2b)
3.5g K2HPO4 +
1.5g KH2PO4 per 100 ml aqua dest.
Polyethylene glycol
PEG 6000, for protoplast transformations (V.2b)
40% solution in 1x SMM (10 g, added to 25 ml with 1 x SMM). Sterilise 10 min, 5 lbs
Serum albumine (BSA)
Used for protoplast transformations; 2% solution
SMM 2 x Used for protoplast transformations; 1.00 M sucrose
0.04 M maleinic acid 0.04 M MgCl2
pH 6.5 (with conc. NaOH)
Make a 500 ml solution, divide it in 50 ml portions and sterilise them. sterilise: 10 min, 5 lbs.
SMMP For protoplast transformations
Mix equal volumes 2x SMM and 4x PAB.
Sodium-succinate For protoplast transformations 1 M (270 g/l), pH 7.3 Spizizens minimal
salts (5x) MZ
Only used in agar media; not in liquid media. Per liter (dissolve in this order):
(NH4)2SO4 10.0 g
K2HPO4 70.0 g
KH2PO4 30.0 g
trisodiumcitrate 5.0 g
MgSO4. 7H2O 1.0 g pH 7.4; This solution is 5x more concentrated as SZ
Spizizens salts SZ Used for minimal growth medium and hunger medium.
5times dilution of MZ (Spizizens minimal salts) in sterile aquadest. Sporulation
medium
Per liter:
8 g dehydrated nutrient broth 0.5 ml 1 N NaOH
10 ml 0.1 M MgSO4 10 ml 10% w/v KCl
Steriliseer 10 min, 15 lbs., and add after that (sterile) 1 ml 1 M Ca(NO3)2
1 ml 0.01 M MnCl2 1 ml 0.001 M FeSO4
Top agars For the plating of bacteriophage's there are different types of top-agar. These agars contain only 0.7% agar and are for the rest identical to the standard agars. Trypton-Yeast
(TY) bouillon (= Luria Broth)
A complete medium suitable for the growing of B. subtilis and E. coli. Also suitable for growing host cultures for phage infections; add in that case MgCl2 upto 10-2 M (or with SPP1, 4 x 10-2 M)
Per liter: trypton 10 g yeast extract 5 g
NaCl 5 g
Na steriliseren:
MnCl2 10 ml from a 10-2 M stock. [MgCI2 can be added later]
adjust pH to pH 7.2 with NaOH. TY medium is sensitive to excessive sterilisation. Trypton-Yeast
(TY)-agar
Complete, non-selective medium. Used for B.subtilis en E.coli after transformation with plasmids. Also suitable for plating bacteriophages.
Same as TY-bouillon, but with the addition of 1.5% minimal agar in stead of adest. Remark : many phages need divalent cations for a good attachment. Supplement for SPP1 with 20 mM and for other phages with 5 mM MgCl2 (end concentration).
Yeast-extract solution 10%
For DM3 agar : 10 g in 100 ml aqua dest. sterilise: 15 min, 5 lbs.
I.5. Media for Lactococcus lactis
Lactococci are micro-organisms lacking metabolicpathway's for the synthesis of some essential cel-components (amino acids, bases). They make high demands on their growth medium. The most used medium is M17-broth. This is made in the kitchen.
All media : sterilise 15 minuten at 15 psi, final pH 6.9 +/-0.2. M17 agar M17 medium supplemented with 1.5% agar M17 medium GM17 medium per liter polypeptone 5.0 g phytopeptone 5.0 g yeast-extract 2.5 g beef-extract 5.0 g ascorbaat 0.5 g β-glycerofosfaat 19 g MgSO4.7H20 (1M) 1 ml
Premixed M17-broth is available: dissolve 37.5 g per liter water.
Take care: M17 is a very rich medium and should therefore be stored without carbon source (to prevent infections). Add just before use :
2.5 ml 20% glucose per 100 ml M 17 !!!!!. After addition of glucose the medium is called GM17
Reconstituted skim milk
Dissolve 10gr Skimmilk-powder in 95 ml demi.Stirr until the skimmilk is dissolved completely. Steam the solution twice during 15'. Dependent on the experiment the solution also can be steamed 10'.
SGGM17 medium The electroporation of L.lactis gives rise to higher transformation efficiencies (High yield electrotransformatie) and requires growing of the cells in SMGG17 medium.
SGGM17 = SGM17 plus 1.0% glycine (MG1363) SGGM17 = SGM17 plus 0.4% glycine (IL1403)
100 ml sterile M17 17 gr Sucrose 1.5 gr Glycine 10', 15psi
SGM17 medium For standard electroporations SGM17medium is used. This is GM17 medium with sucrose (improves the survival of the cells)
SGM17 = GM17 medium + 0.5 M sucrose Whey-based
medium
a) Dissolve 5gr Whey-powder in 90 ml(endvol) demi b) Dissolve 1gr Casitone in 10 ml (endvol) demi(10%)
c) Dissolve 60gr Na-β-glyceroposphate in 100 ml (endvol) demi (60%) d) Sterilise the 3 solutions ( The Whey solution will be turbid after sterilisation!) e) Add to the Whey solution:
- 1 ml 10% Casitone opl.
- 3.3 ml 60 % Na-β-glyceroposphate opl. - 2.5 ml 20% glucose opl.(als de stam lac- is).
The final concentrations are: 5% whey, 0.1% casitone, 2% Na-β-glyceroposphate and 0.5% glucose.
Technical Manual II
Common basic methods
II.1 BACTERIAL- AND BACTERIOPHAGE STRAINS 18
1.1 Starting material, pure culture 18
1.2 Storage of strains 18
II.2 WORKING UNDER STERILE CONDITIONS 19
II.3 STERILISATION; MELTING AGAR(OSE) 19
3.1 Pression cooker 19
3.2 Magnetron 20
II.4 STERILISATION WITH THE HELP OF MEMBRANE FILTERS 20
II.5 PIPETTING 21
5.1 Glass pipettes (for standard dilution’s, plating etc.) 21
5.2 Micropipettes 21
II.6 DILUTION SERIES 22
II.7 POURING AND DRYING AGAR PLATES 22
II.8 PLATING OUT BACTERIA AND BACTERIOPHAGES 23
8.1 B. subtilis and E. coli 23
8.2 Protoplasts 23
8.3 Transferring colonies to test plates by tooth picking 23
8.4 Bacteriophages (of B. subtilis) 24
II.9 CENTRIFUGING AND WASHING BACTERIAL CELLS 25
II.10 DIALYSING 25
II.11 WASHING OF PHENOL 26
II.1 BACTERIAL- AND BACTERIOPHAGE STRAINS
1.1 Starting material, pure culture
Most experiments start out with making pure cultures of several strains. Keep in mind that the strains in the freezer may not be right; they can be contaminated with other bacteria, and reverse mutations (reversions) can occur. Therefore it is wise to have a so called entplate of each strain (see II.1.2.a1), on which the bacteria or phage suspension is being plated out, in a dilution that single colonies/plaques can be distinguished. Use such a single colony or plaque as a starting material. Usually the genotypes have to be checked. Entplates can be stored for a short period (several days to weeks). Don’t wait too long making new entplates. Keep entplates with B.subtilis at room temperature and with
E.coli or phages in the refrigerator.
1.2 Storage of strains
a. B.subtilis For daily use
1. Entplate When during a series of experiments the same strains are being used, it is best to make entplates with separate colonies and refresh these every 1 to 3 weeks (see above).
2. Freezer culture It is also possible to freeze (-80°C) cultures with 20% glycerol (sterile) in small portions and start cultures for experiments from here. For important strains: make two tubes and place them in different freezers (spread of risks).
For (semi)permanent storage
1. Freezer culture Add 10 to 20% (w/v) sterile glycerol to a pure culture. Then freeze the culture at -80°C in at least two freezers (spread of risks!).
2. Freeze drying A good method, but cannot be applied because we do not have the equipment.
3. Sporulation medium On sporulation medium sporulating Bacillus strains can be stored for years. Make the agar medium in well sealed tubes or flasks. Store at room
temperature. Per litre:
- 8 g dehydrated Difco nutrient broth - 0,5 ml 1 N NaOH
- 10 ml 0,1 M MgSO4 - 10 ml 10% w/v KCl - agar till 1,2%
Sterilise, 10 min, 15 Lbs, then add (sterile): - 1 ml 1 M Ca(NO3)2
- 1 ml 0,01 M MnCl2 - 1 ml 0,001 M FeSO4
b. E.coli For daily use
1. Entplate Usually on TY-agar (plus antibiotics if necessary). 2. Freezer culture As for B.subtilis (see above, 1.2.a2)
For (semi)permanent storage
c. Bacteriophages For daily use
1. Sterile filtrates Make a lysate, preferably from a single plaque (from a fresh entplate). Filtrate by means of a membrane filter and store the lysate in the refrigerator. 2. Entplate For preparation of the lysates (1) use a single plaque from a fresh entplate. For (semi)permanent storage
1. Sterile filtrates of phage lysate’s
As in c.1, after adding 1/10 volume part dimethyl sulfoxide (DMSO). 2. Freezer lysate Freeze the lysate (c.1) in 10 to 20% (v/v) glycerol (-80°C).
II.2 Working under sterile conditions
Working sterile is very important when doing research with micro organisms. Therefore minimise the chance of contamination. This usually means using the standard microbiological techniques for working sterile. During the course it will become clear that for a number of standard techniques (for instance pouring and drying plates, handling pipettes and plating out bacteria and bacteriophages), the microbiological rules for working sterile cannot be carried out strictly. The size of the experiment and the period of time in which certain actions have to be done (for example many plates) can force us to make a compromise between demands to working sterile and demands to the tempo of working. So keep asking yourself how important working sterile for a certain action is.
Many early actions in an experiment demand a high discipline with regard to working sterile (e.g. starting cultures) while in later stages (e.g. plating out) a possible contaminant hardly interferes with the experiment.
Frequently made mistakes:
o Stock solutions are not sterile (casamino acids, glucose and growth factors are notorious examples. Refresh regularly, or sterilise!)
o Overnight cultures are contaminated.
o Pipettes are not handled sterile (don’t touch the tip).
o Bottles in which or from which is being pipetted are open too long.
Note that the distributed glass ware is sterilised when it is shut with a cap (bottles and tubes).
Note: to prevent confusion, students in the practical course also have to remove caps from the glassware they have used. Please get accustomed to this habit.
II.3 Sterilisation; melting agar(ose)
3.1 Pression cooker
1. Unscrew the caps of the bottles a little bit (to let over pressure escape) and put the bottles in the pan.
2. Fill the pan with at least 2 cm demi water, or, for bottles of 1 litre, till at least half the height of the bottle (to prevent breaking during sterilisation).
3. Place the lid (without pressure weight!) on the pan. Point the arrow on the outside of the lid at the word Open on the upper part of the pan. Lock the lid by turning it a bit to the right.
4. Light the gas and wait until all air in the pan has been replaced by steam (the steam escapes through the valve).
5. Now place the pressure weight on the valve and wait until the weight starts to swing as a consequence of the escaping steam.
6. Take back gas until the pressure is stable on 15 lbws and maintain this during the sterilisation time.
7. Turn off the gas and wait till the over pressure is gone. Then take away the pressure weight of the valve.
Note: Be careful with agar or solutions just after sterilisation, delay of boiling! Don’t hold your face above the bottles.
Directives for sterilisation times
agar, all kinds 15 min 15 lbs
bouillon, all kinds 15 min 15 lbs
solutions of inorganic salts 15 min 15 lbs
dry glass ware 15 min 15 lbs
amino acids, bases and vitamins 30 min steam
glucose and other sugars 10 min 5 lbs
Penassay broth (PAB) 15 min 5 lbs
yeast-extract 15 min 5 lbs
2 x SMM 10 min 5 lbs
agarose solutions 10 min 15 lbs
Eppendorf cups 20 min steam
pipette tips 20 min steam
Remark Sensitive (bio)chemicals and antibiotics are usually being sterilised using
membrane filters (III.4). Plastics and synthetic materials ( tips for micropipettes; Eppendorf cups): can also be sterilised dry (minimal 2 hr using the dry autoclave in the kitchen).
3.2 Magnetron
1. Use only bottles with plastic caps . 2. Remove the caps from bottles.
3. Sterilisation time depends on the used volumes. 4. Take care: boiling over occurs fast
II.4 Sterilisation with the help of membrane filters
The sterilisation with the help of membrane filters is often used in the case of phage lysates and heat labile material. Before filtration remove the crude parts like cells by centrifugation . Sterile disposable filter holders are available in pore sizes of 0,20, 0,45 en 0,80 µm .Filter diameters: 25 and 47 mm.
Method:
1. Fill a sterile syringe (without needle) and attach it to the filter. 2. Push the fluid through the filter using the syringe.
II.5 Pipetting
5.1 Glass pipettes (for standard dilution’s, plating etc.)
During pipetting mistakes are made often. Here are some practical hints: o Take notice of the correct volume of the pipette:
0,1 ml: white or green 1,0 ml: yellow
10,0ml: brown
o To all pipettes applies that the scale goes from high to low and ends in the point. o Never grasp a pipette that you are going to use at the point.
o After sucking up, and blowing liquids out of the pipettes, roll the point of the pipette against the glass wall at an angle of 45°, to remove drops of liquid.
o Blow the content of the pipettes away under liquid level.
o Mix every time after pipetting the content of the tubes by shaking o Take a new tip for every dilution step.
o Series of platings can be done with one tip, if one starts at the maximum dilution.
5.2 Micropipettes
Nowadays many acts of pipetting are being done on microscale, especially for manipulating with DNA and proteins. In a growing extend bacteria are being plated out with micropipettes. The department mainly uses pipettes of the trademarks Oxford, Gilson and Eppendorf:
Gilson series:
2 - 20 µl P20 variable, yellow (or white) tips 20 - 200 µl P200 variable, yellow (or white) tips 100 - 1000 µl P1000 variable, blue tips
Eppendorf:
0,5 - 10 µl type 4710 variable, thin white tips
Sterile tips In many cases it’s necessary to work with sterile tips, for instance when pipetting restriction enzymes and ligase from central stocks, and for plating out bacteria. Sterilise the tips as follows: 2 hours in the dry autoclave or steam 20 min at 100øC.
Clean tips To keep the supplies of tips clean, "pin" the tips with your pipette from the box with tips. Then press the tips on the pipette. Never touch the tips with your hands! (you can use plastic gloves!).
Indications for use
1. Use plastic gloves and wear a lab coat when pipetting enzymes from stocks (restrictions, ligations, etc.). This is obligatory!
2. Adjust to desired volume: press down piston, turn adjust ring till desired state. 3. Place the tip on the pipette (see above).
4. Press piston till first resistance.
5. Place the tip in the liquid from which to pipette. 6. Let the piston come up calmly.
8. Press the liquid into a tube by pressing through the first resistance. Keep the tip to the wall of the tube.
9. Check whether the liquid was transferred indeed.
When using an Eppendorf tube ("Epjes") and pipetting small volumes, apply the liquid just under the upper part of the tube and spin down (few seconds, Eppendorf) . Then mix.
Important: Never use a dirty tip for pipetting enzymes
II.6 Dilution series
When plating out an undiluted bacterial culture or a phage suspension, usually so many colonies (plaques) appear on the plate, that they are no longer separate. Diluting is the solution.
Dilution with factor 10 Pipette 0,1 ml suspension in 0,9 ml dilution medium (for B. subtilis after transformations with chromosomal DNA and phages: hunger medium, other wise TY)
Dilution with factor 100 Pipette 0,05 ml suspension in 4,95 ml dilution medium
Dilution with factor 10 5 If we want for instance dilute with a factor 105 , we can do this in several ways:
1. Pipette 0,1 ml in 0,9 ml dilution medium (10-1). Mix. Pipette from there
again 0,1 ml in a next tube with 0,9 ml dilution medium etc. The dilution scheme is as follows:
0,1 0,1 0,1 0,1 0,1 (10-5)
0,9 0,9 0,9 0,9 0,9
2. According to the scheme:
0,05 0,05 0,1 (10-5)
4,95 4,95 0,9
Of course other schemes are possible, e.g.:
0,10 (2.10-2) 0,05 (2.10-4 ) 0,1 (2.10-5)
4,90 4,95 0,9
Using a micropipette (use sterile tips!) even larger dilution steps are possible. For instance 5 µl in 5 ml dilution medium (10-3).
II.7 Pouring and drying agar plates
Pouring After melting agar (supplement needed if using minimal agar: minimal salts, glucose and growth factors (III.B.9)). Let the agar cool till 50 à 70°C and then pour the plates (± 20 ml/plate). Don’t forget to add selective antibiotics and/or X-gal and IPTG.
Note: Do not pour and use plates that contain riboflavin by full sunlight; the riboflavin
will be inactivated..
Drying A short while before use the plates have to be dried in the warm room. Put them opened upside down and as tiles in the shelves.
Drying time for plating out 0,1 ml : 30-45 min Drying time for plating out 0,2 ml : 60 min
Some remarks
- Never add antibiotics to hot agar, first cool down till ±60°C
- Plates with antibiotics should not be older than 1 week. Make sure that stock solutions antibiotics are not too old (several weeks). They can be stored in the freezer in small portions.
II.8 Plating out bacteria and bacteriophages
8.1 B. subtilis and E. coli
Use for dilution the kind of medium in which the bacteria were grown, for instance hunger medium for competent B. subtilis (when transformed with chromosomal DNA), and TY bouillon in many other cases.
0. Sterilise the bars used to spread bacteria as follows: . dip in alcohol and burn off the alcohol;
. let the bar stand for at least one minute.
Be careful burning off the alcohol; bottles with alcohol easily catch fire! Don’t panic, but place calmly a lid (for instance from a petri dish) on the burning bottle. The fire will extinguish almost at once.
1. Pipette 0,1 ml of the right dilution on the plate. Touch the plate with the pipette point while blowing out the sample.
2. Spread the liquid quickly after, without pressure, homogeneously over the plate.
3. After plating out incubate the plates upside down (lid down) at 37°C (incubator or warm room). Other methods for plating out
1 Spread the suspension homogeneously over the pla te with the point of a glass pipette.
2 Apply ± 8 glass balls (sterile, diameter ± 4 mm) to the plate and spread the suspension by shaking firmly.
Don’t use this method for protoplasts.
8.2 Protoplasts
1 Dilute in 1 x SMM
2 Plate out with micropipettes
3 Spread, without pressure, with a bar.
8.3 Transferring colonies to test plates by tooth picking
It can be nessecary to transfer colonies to another agarplate for testing the presence of (plasmid-encoded) antibiotic-resistance(s) for instance when using marker inactivation for cloning or in experiments on stable maintenance of plasmids. Often transferring is done with tooth pickers. It’s convenient to divide the plate in compartments. This can be done by placing a grid with 52, or 120 numbered squares (see the back of this manual). The position of the colony on the plate corresponds with a certain number.
Procedure
1. Sterilise in a beaker of 100 ml a number of tooth pickers (10 min, 15 lbs, pressure cooker). Close the beaker before sterilising with aluminium foil.
2. Take with a tweezers (sterilised by taking the point through the flame) a sterile tooth picker from the stock just touch a colony. You can hold the tooth picker on the back side with your hands.
3. Draw a marker line on the edge of the plate to which the colony has to be transferred (on the bottom) for orientation towards the numbers on the grid. Put the grid under the new plates with the marker line over the first numbers.
4. Now transfer the colony by touching the new plate with the tooth picker with bacteria in a compartment. With one tooth picker two to three plates can be done without touching the "mother" colony again. To check that you did touch a mother colony usually the colony is also transferred to a plate without any agar.
8.4 Bacteriophages (of B. subtilis)
This chapter is specialised in bacteriophages of B. subtilis, because the department works mostly with these phages. For E. coli phages (e.g. lambda and M13), see Molecular cloning (Editors: Maniatis et al., 1982 en 1989).
1. Dilution and plating out; phage titers
Phages of B. subtilis are diluted in hunger medium (III.B.4) Bacteriophage should be plated together with the host bacteria (so called indicator bacteria). (See for indicator cells: II.8.d2). Apply the indicator in a concentration that confluent growth occurs (the plate becomes overgrown with a layer of cells). Where the phage particles fall phage multiplication occurs, visible as a lysis spot (plaque). By counting the plaques the amount of active phage per ml can be determined (titer determination). Most phages can be plated onto rich agar media. For SPP1 and H1 TY agar (+10 mM MgCl2) is suitable.
Method The standard procedure uses topagars (see also alternative
possibility for SPP1, as described later).
o Melt the concerning topagar and place at 44°C (water bath).
o Pipette portions of 2,5 ml in small tubes and place at 44°C. o Dilute the phage and pipette 0,1 ml in a tube with topagar. o Add immediately 2-3 drops (± 0,1 ml) indicator culture and mix (role tube between hands).
o Pour content over the plate fast and spread the top layer homogeneously over the plate by turning over the plate a bit by hand.
o Do not move the plates until the top layers are congealed. o Incubate the plates in the warm room (or incubator). Alternative method for
SPP1
This method does not use topagar and is therefore faster (the method might be used for other phages, but the plaques are finer using the topagar method).
o Dilute the phage and pipette 0,1 ml on the plate.
o Spread the mixture homogeneously over the plate with a sterile bacteria spreader, as done plating out B.subtilis (IV.8.a).
2. Indicator cells Most phages yield the best results on physiological active cells in the
exponential growth phase. SPP1 is an exception: this phage yields good plaques on old (overnight culture) cells.
Procedure General o Grow 50 ml overnight culture in a suitable medium.
o Dilute ± 1 hour before plating 1:10 in fresh medium and grow the cells at 37°C with moderate aeration.
SPP1 o 50 ml overnight culture in TY bouillon + 10-2 M Mg Cl 2 o Dilute just before use cells 1:2 with TY-bouillon.
N.B. For regular use it’s convenient to have a stock of indicator cells in the freezer. Add glycerol tot 10-20% v/v to the overnight culture and freeze portions of e.g. 1 ml. Dilute just before use 1:2 with TY bouillon.
Remark Competent B.subtilis cultures are also suitable as indicator. Dilute before use 1:3 with TY bouillon.
Suitable strains: 1-30 (MCB) for SPP1 1G-20 for other phages
II.9 Centrifuging and washing bacterial cells
Necessary when collecting cells, or replacing the medium. For instance to get rid of toxic substances or certain nutrients.
Procedure
1. Centrifuge the cell suspension during 30 sec in the Eppendorf centrifuge; 10 min at 6 k in a High-speed centrifuge; or 10 min in the table centrifuge at maximum High-speed (ór in transformation bottles at maximum state 5).
2. Pour off the liquid carefully, don’t loose cells.
3. Optional: resuspend the cells in the wash medium and centrifuge again. Repeat pouring off and resuspend the cells in the new medium.
II.10 Dialysing
When dialysing nucleic acids or proteins it’s important to keep the hose clean (for instance remove two valid ions) and sterilise as well as possible. This can have great impact on the quality and storage life of the preparations.
Procedure
General Use plastic gloves when handling the hose and applying and removing samples
Treatment hose 1. Cut off a piece of hose of the right length and size.
2. Rinse (inside and outside) of the hose several times with warm tap water and then with aqua dest.
3. Cook the hose ± 10 min in 10-3 M EDTA. Always use "fresh cooked"
hose.
Dialyse 1. Put one or more knots (solid) or dialyse-clip (fastener) on one end of the hose.
2.Apply sample with a pipette, or micropipette. 3. Remove as much as possible air above the sample.
4.Clamp the other end of the dialyse hose with a orange clip. Take care that in high-osmotic solutions the liquid does not take more than half the volume in the hose.
5. Put the hose in the dialyse liquid and stir with a magnet stirrer. Usually the dialyse liquid has to be refreshed several times during the procedure. 6. After dialysing: cut (with clean scissors) the hose and remove liquid with a
micropipette; ór push sample in a tube or bottle (use gloves!).
Suggestions for dialysing time temperature number of
refreshments DNA (removing phenol etc.): 24 hours cold room 3x1 litre SSC or T1OE1
removing CsCl from bacteriophage and DNA:
2 hours room temp. 3x500 ml SSC, or T1OE1
changing buffer/desalting of proteins: 16 hours cold room 3x1 litre
II.11 Washing of phenol
For isolating of qualitative good DNA it’s necessary to use freshly washed or destilled phenol, from which peroxides and other oxidising chemicals are removed.
Warning Phenol is a toxic chemical. Do not breathe the damp; When spoiling on the skin wash immediately with water and soap (painful blathers). When spoiling on the floor or table clean phenol instantly. Work in a fume hood; wear gloves and always use a pipetting balloon. Put phenol waste in the right waste box!
Wash procedure
1. Mix 1 kg phenol with 200 ml 2 M Tris.HCl pH 7.4 and 260 ml water. 2. Let stand 10-20 hours at 20°C (everything will then solve).3. Remove water phase.
4. Add : 100 ml 2 M Tris.HCl pH 7.4. 1 ml β-mercapto ethanol. 500 mg 8-hydroxyquinoline. 5. 5 months storable in the dark at 20°C.
When the phenol has to be kept longer, then freeze at -20°C while the hydroxyquinoline is yellow and not orange.
When pipetting phenol: in defrosted state water gathers in the upper layer, the phenol is in de layer beneath. Use pipetting balloon, or micropipette when working with phenol.
Technical Manual III
Transformations and transfections
III.1 TRANSFORMATION OF COMPETENT B. SUBTILIS 28
1.2 Competent culture 28
1.3 Transformations 28
1.4 Transfections 29
1.5 Transformation protocol A from F.Kunst 29
III.2 PROTOPLAST TRANSFORMATION (B. SUBTILIS) 30
2.1 Protoplast Transformation in general 30
2.2 Media 30
2.3 DM3 agar 31
2.4 Protoplast-generatiom 31
2.5 Transformation 32
III.3 TRANSFORMATION OF E. COLI WITH PLASMID DNA 32
3.1 Strains 32
3.2 Competent culture CaCl2 method 32
3.3 Transformation 33
III.4. ELECTRO TRANSFORMATION OF E. COLI 33
4.1 Preparation of cells 33
4.2 Protocol for Electro-transformation using the BioRad Gene Pulser 33
III.5. ELECTRO TRANSFORMATION OF LACTOCOCCUS LACTIS 34
5.1 Preparation of cells 34
5.2 Electroporation 34
III.1 Transformation of competent B. subtilis
1.1 Over night cultures Grow over night cultures during16-18 hours at 37°C.
1. Inoculate 10 ml minimal growth medium (III.B.1; 100 ml flask) using a öse (or the tip of a sterile pipette) with a part of a colony from a culture plate or with ± 0,05 ml frozen stock (-80°C).
2. Incubate in a shaking water bath at 37°C, 200 RPM.
1.2 Competent culture
1. Add 1,3 ml of the over night culture to10 ml minimal growth medium in a 100 ml flask, (III.B.1), making the A450 ± 0,5.2. Grow for 3.15 hours at 37°C (shaking water bath, 37°C, 200 RPM). The A450 will be between 1,2 - 1,6.
3. Dilute 1:1 with starvation medium (III.B.4) and incubate (37°C) again 2.00 hours (shaking water bath , 37°C, 300 RPM)
4. The culture is now maximal competent and stays for ± 1 hour fairly competent (keep shaking!)
Freezing
competent cells
If desired the competent culture can be frozen for future use :
1. Add sterile glycerol to10% (v/v), mix well and distribute over 0,1 to 2 ml portions (sterile tubes).
2. Place the tubes at -80°C.
3. Use a fresh tube for each experiment (don’t freeze what is left over) and place the tube just before the experiment in a 37°C water bath. 4. Use the culture immediately after defrosting.
1.3 Transformations
With chromosomal DNATransformations with saturated DNA are done as follows: 1. Mix 0,45 ml competent cells with 0,05 ml DNA à 2µg/ml SSC
(saturation grade ± 1.0 µg/ml).
2. Incubate during 45 min at 37°C in a shaking water bath (200-RPM). If necessary stop further uptake of DNA with DNAase (to 20 µg/ml, 10-2 M Mg++; 5 min at 37°C)
3. Plate out on selective minimal agar (III.B.9; IV.8.a). The procedure can be changed depending on the experimental conditions (e.g. volumes)
With plasmid DNA Transformations with plasmid DNA are mostly done on micro scale e.g.:
1. Mix 10 µl DNA (in H2O; 0,1 to 0,5 µg DNA if saturation is needed) with 500 µl competent cells in an Eppendorf cup of 2,5 ml.
2. Place for 30 min at 37°C in a shaking water bath (200 RPM). 3. In most cases it is necessary to let the resistance come to expression
*, if selection for antibiotics is done. Add 300 µl TY bouillon. 4. Incubate again 30-45 min at 37°C (shaking water bath, 200 RPM). 5. Plate out 100, 10-1 (and sometimes 10-2) on MM agar + desired
antibiotics.
* Selection on resistance against chloramphenicol needs no expression time. Plating out can be done directly after transformation.
1.4 Transfections
Are done in the same way as Transformations (see c), in this case with bacteriophage DNA.1. Mix 50 µl phage DNA (1 à 4 µg) and 450 µl competent cells (or change proportionally)
2. Incubate 30 min at 37°C (shaking water bath, 200 RPM).
3. Stop further DNA uptake if necessary with 100 µl DNAase (stock: 40
µg/ml, in 5 x 10-2 MgCl
2); incubate 5 min at 37°C. 4. Plate out with indicator bacteria, as shown in IV.8.d.
Remark: under optimal conditions you will receive ±5 x 105
transfectants / ml with SPP1 DNA. Other phage DNA's are less efficient.
1.5 Transformation protocol A from F.Kunst
Media Phosphate-citrate buffer (10 x PC)
10 x concentrated stock solution contains per litre : K2H PO4 (anhydrous) 107 g
KH2 PO4 (anhydrous) 60 g
Trisodium citrate (5 H2O) 10 g
Dilute the stock solution and check the pH of the 1xPC-buffer. Adjust (if necessary) to pH=7. (1xPC corresponds with SZ without ammonium sulphate)
10 ml MD medium :
1 x PC buffer 9.20 ml
Glucose (50 %, w/v) 0.40 ml
L-tryptophan (5 mg/ml) 0.10 ml
Ferric ammonium citrate (2.2 mg/ml) 0.05 ml
Potassium aspartate (100 mg/ml) * 0.25 ml 1 M MgSO4 0.03 ml
10.0 ml
* Potassium glutamate can also be used but is somewhat less efficient. Add0.2 ml 5 % casein hydrolysate to 10 ml MD; this medium is called MDCH.
0. Make a 2 ml ON culture at room temperature in LM broth (LB medium supplemented with growth factors for auxotrophe mutants and 3mM MgSO4).
1. Make a dilution of this ON culture to OD600 ~ 0.05 in 5 ml MDCH medium and grow at 37° C in a shaking water bath to T0 (transition of exponential to stationary phase)
2. Add 1 fresh MD medium (without casein hydrolysate.) and grow for 1 hour in a shaking water bath at 37° C.
3. Add DNA and grow during 20 minutes in a shaking water bath at 37° C. 4. If there is selection on antibiotics it will be necessary in most cases to
express the resistance before plating. Add 300 µl TY bouillon. 5. Incubate again 30-45 min at 37°C (shaking water bath, 200 RPM). References for this protocol :
Kunst F., Msadek T. and Rapoport G. (1994). Signal transduction network controlling degradative enzyme synthesis and competence in Bacillus subtilis. In Regulation of Bacterial differentiation, P.J. Piggot, C.P. Moran Jr. and P. Yougman (ed.), pp 1 - 20.
Kunst F. and Rapoport G. (1995). Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J. Bacteriol., 177: 2403-2407.
New
transformation protocol B
1. Make a 2-ml ON preculture at room temperature in LM broth (LB medium supplemented with growth factors for auxotrophe mutants and 3mM MgSO4).
2. Make a second preculture by dilution of this ON culture to OD600 ~ 0.2 in 5 ml LM broth.
3. Incubate in shaking water bath at 37°C to OD600 ~ 1.
4. Dilute 0.5 ml of this second preculture in 10 ml MD medium (dilute immediately; introduce no centrifugation / resuspension step!) (The first preculture can also be used but the transformation frequencies will be most likely lower if the first preculture is already in the stationary phase.)
5. Incubate in shaking water bath at 37°C during 4 hour to reach the stationary phase.
6. Add DNA one hour after T0 (OD600 ~ 1 - 1.5), incubate in shaking water bath at 37°C during 20 minutes.
7. If there is selection on antibiotics it will be necessary in most cases to express the resistance before plating. Add 300 µl TY bouillon. 8. Incubate again 30-45 min at 37°C (shaking water bath, 200 RPM).
III.2 Protoplast Transformation (B. subtilis)
2.1 Protoplast
Transformation in
general
In Transformations of competent B.subtilis cells only multimeric plasmid molecules are active. This is one of the reasons why transformation percentages are low (10-3 to 10-4), and shotgun cloning have low efficiencies. Protoplasts can be transformed with monomeric plasmids and for that reason can give some advantages to find recombinant clones. Disadvantages of protoplast transformation are: the procedure is very laborious and there is a low efficiency of transformation with bigger (> 6 kb) plasmids. Transformation percentages up to 10% of regenerated protoplasts can be obtained.
2.2 Media
2 x SMM 1 M sucrose
0,04 M maleïnicacid 0,04 M MgCl2
Adjust pH to 6.5 with conc. NaOH-solution. Sterilise: 10 min 5 lbs Make 500-ml solution and divide in50 ml portions before sterilisation. 4 x PAB 7 g Bacto Penassay Broth per 100 ml.
SMPP Mix equal volumes 2 x SMM and 4 x PAB. PEG 40% 10 g polyethylene glycol 6000 in 25 ml 1 x SMM;
Sterilise 10 min, 5 lbs.
Agar 4% solution in water
Na-succinaat 1 M (135 g/500 ml, pH 7,3)
phosphate-sol. 3,5 g K2HPO4 and 1,5 g KH2PO4 per 100 ml.
MgCl2 1 M
yeast extract Difco, 10%, sterilise 15 min, 5 lbs.
Glucose 20%, sterilise 10 min, 5 lbs.
Lysozyme 20 mg/ml in SMMP, filter sterilize serum albumin
(BSA)
2%, bovine serum albumin, filter sterilise!
All media should be made separately. Sterilise 15 min, 15 lbs., unless otherwise stated.
2.3 DM3 agar
Protoplasts are regenerated on DM3 agar. This takes 2 to 3 days. The efficiency of regeneration varies from ± 1 - 20%.For ± 20 plates:
1. Mix (in bottle of 500 ml):
Na-succinate(1M) 250 ml casamino acids(5%) 50 ml yeast extract (10%) 25 ml phosphate solution 50 ml glucose (20%) 15 ml MgCl2 (1 M) 10 ml BSA (2%) 2,5 ml
2. Heat to ± 60°C and add100 ml autoclaved agar (4%). If desired add antibiotics : chloramphenicol to 10 µg/ml, of kanamycine to 150 µg/ml. 3. Mix gently and pour de platen.
2.4
Protoplast-generatiom
It is of great importance to use clean glassware or plastic. Protoplasts are extremely sensitive for detergents! 1. Start an overnight culture in PAB (1*) (usually 10ml) 2. Dilute o.n. culture 1:10 in 10 or 20ml PAB (Volume X) 3. Incubate 2 hrs 37°C; 200 rpm until A450 is 1.5 - 2.0
(OD is not really critical, just an indication)
4. Centrifuge sterile 5 min 8000 rpm, in a 50 ml Greiner tube (blue cap) 5. Resuspend in 2.5 - 5ml SMMP (vortex may be used at this step)
6. Add 1.5 mg/ml Lysozyme
7. Incubate at 37°C, shake in Greiner tube. Note; it will not work if you incubate in 2ml tubes. Believe me, I tried several times. Incubation in a Greiner tube is the best method.
8. Follow protoplast formation under microscope. First are visible after 30 min.; protoplasts are globular, cells are rods. When using a Greiner tube, all cells are usually protoplasts within 30 minutes. Using 2ml tubes it typically takes much longer (up to 2 hours) and the cells will not be competent.
9. When less than 1% of the cells are rods, or after 2hrs; centrifuge 5' 5000 rpm (do not wait longer than 2 hrs)
10. Resuspend pellet in 1 or 2ml SMMP (0.1 x Volume X); be careful protoplasts are vulnerable.
2.5 Transformation
1. Mix in a sterile plastic tube ± 0,2 µg DNA (volume max.10 µl; if not bring the DNA solution to 1 x SMM) and 150 µl protoplasts.2. Add 450 µl PEG-6000 sol.; mix well. 3. 2 min room temperature.
4. Add1,5 ml SMMP, mix.
5. Centrifugate 10 min at 5, table centrifuge (or 2 min at10, Eppendorf) 6. Discard supernatant as much as possible (decant).
7. Resuspend pellet in 150 µl SMMP.
8. Incubate 90 min, 37°C, 100 RPM (expression of resistance). 9. Make if necessary dilutions in SMM.
10. Plate out:
10-4 on DM3 without antibiotics (regeneration)
100, 10-1, (10-2) on DM3 with antibiotics
Be as gently as possible while spreading out the protoplasts over the plates Plate out on TY-agar at 10-2 to determine the amount of not protoplasted cells.
III.3 Transformation of E. coli with plasmid DNA
3.1 Strains
HB101 RecA mutant, some features: hsdM, hsdR, supE44, endA. Suitable for plasmid transformations.
C600 rec+; good recipient for transformations with plasmids.
JM83 Good for transformations with lacZ plasmids. Contain a deletion in the JM101 LacZ gene on the chromosome.
BHB2600 Suitable intermediate host for cloning in B. subtilis. MC1061 Suitable for electro transformations.
GM48 Is missing methylatingsystems for adenine (dam-) and cytosine (dcm-). For this reason it is a suitable host for obtaining completely unmethylated plasmid DNA. The transformability of this strain is low.
NM522 Used as host for blue-white screening with pBlueScript
3.2 Competent culture
CaCl2 method
1. Grow an overnight culture in 10 ml TY in a shaking water bath 2. Dilute 1:100 in fresh TY.(1ml is sufficient for 1 transformation ) 3. Grow at 37°C in a shaking water bath to A600 = 0,3 (about 1,5 hour). 4. Centrifugate 10 min 4°C, 5000 RPM, or in an Eppendorf centrifuge 30
sec (not longer); decant supernatant.
5. Resuspend pellet in ice-cold (0°C) 0,1 M CaCl2, in half the volume used in the dilution step(2) . The CaCl2 should be fresh, maximal 1-2 weeks.
6. Store on ice, 30 min (may longer).
7. Centrifugate 10 min at 5000 RPM, 4°C, or in an Eppendorf centrifuge 30 sec (not longer).
"competent" pellet is much cloudier and there is a hole in the pellet which indicates already good competence
8. Resuspend the pellet with the use of an micro-pipette in ice-cold 0,1M CaCl2, 1/10 of the volume used in the dilution step(2).
9. The cells are now competent and will stay competent for about ± 0,5 hour.
10. You can freeze the cells 10% glycerol, -80°C but because the procedure is very easy you better make fresh competen.
3.3 Transformation
1. Mix ± 0,1 µg DNA in 1 to 25 µl with 100 µl competent cells. 2. 30 min, 0°C (ice)3. Heat shock : 5 min, 37°C. 4. Dilute with 400 µl TY bouillon.
5. Grow 30-60 min at 37°C, 200 RPM.( expression time) 6. Plate out on TY agar + antibiotics. 100 µl per plate
This procedure will give ± 106 transformants /µg DNA (with ccc
plasmid DNA)
III.4. Electro transformation of E. coli
4.1 Preparation of cells
1. Inoculate 100 ml of TY medium with 1/100 volume of fresh overnight culture in TY medium
2. Grow cells at 37°C with vigorous shaking to a OD600 of 0.5 to 0.8 (the best results are obtained with cells that are growing rapidly; the appropriate cell density, there fore, depends on the strain and growth conditions.
3. To harvest, chill the flask on ice for 15 or 30 minutes, and centrifuge in a cold rotor at 5000 x gmax for 10 minutes.
4. Resuspend pellets in a total of 100 ml of cold water. Centrifuge 6000g,20' 5. Resuspend in 50 ml of cold water. Centrifuge 6000g,25'
6. Resuspend in 50 ml of cold water. Centrifuge 7000g,15' 7. Resuspend in 2 ml 10% glycerol. Centrifuge Eppendorf, 1'.
8. Resuspend to a final volume of 0.2 ml in 10% glycerol. The cell concentration should be at least 3 x 1010 cells/ml.
9. This suspension may be frozen in aliquots on dry ice, and stored at -70°C. The cells are good for at least 6 months under these conditions.
Dose Response Curve
tfm
[DNA] µg/ml
Verzadiging bij 0,1 - 1,0 µg DNA per ml
4.2 Protocol for Electro-transformation using the BioRad Gene Pulser
1. Chill the cuvettes and the sliding cuvette holder on ice.2. Set the Gene Pulser apparatus to the 25 µF capacitor, and set the Pulse Controller to 200 Ohm.
3. Gently thaw the cells at room temperature and place them on ice.
4. To a cold, 1.5 ml polypropylene tube, add 40 µl of the cell suspension and 1 to 5µl of DNA in a low ionic strength buffer (such as T10E1). Mix well and let sit on ice ± 1 minute.
5. Transfer the mixture of cells and DNA to a cold, 0.2 cm, electroporation cuvette, and shake the suspension to the bottom of the cuvette.
6. Apply one pulse at the above settings. This should result in a pulse of 12.5 kV/cm with a time constant of 4.5 to 5.0 msec.
7. Immediately add 1 ml of SOC medium* (at room temperature) to the cuvette, and gently but quickly resuspend the cells with a pasteur pipette.
* SOC contains per liter:
20 g Bacto tryptone, 5 g Bacto yeast extract, 0.58 g NaCl; 0.189 g KCl; 2.03 g MgCl2; 2.46 g MgSO4, 20 ml glucose (from 20% stock solution, kitchen) 8. Transfer the cell suspension to a 2ml eppendorf cup and incubate at 37°C for 1
hour in a waterbath.
9. Plate the appropriate aliquots on selective medium.
III.5. Electro Transformation of Lactococcus lactis
5.1 Preparation of cells
- 10 ml O.N. culture (SMGG) in 100 ml SMGG. - Grow to OD600 = 0.2-0.7.
- Wash three times with 50 ml icecold wash buffer - Resuspend in 1 ml wash buffer.
5.2 Electroporation
- 1 ul DNA in 40 ul cell-suspension in ice cold cuvette. - Electroporate at 2.5 kV, 25 uF, 200 Ohm.
- Add 4 ml SMG17MC.
- Incubate two hours. (Ery induction = 50 ng/ml) - Concentrate cells to 0.5 ml.
- Plate on GSM17-agar.
5.3 Buffers
Wash buffer 0.5 M Sucrose(17 g/100ml) + 10% glycerol (11.5ml /100ml). SMGG M17 + 0.5M Sucrose + 0.5% Glucose + 1% Glycine.
SMG17MC M17 + 0.5M Sucrose + 0.5% Glucose + 20mM MgCl2 + 2mM CaCl2. GSM17-agar M17-agar + 0.5M Sucrose + 0.5% Glucose.
Technical Manual IV
DNA isolations
IV .1 CHROMOSOMAL DNA 38
1.1 Chromosomal DNA for transformations, not for enzyme reactions 38
1.2 Chromosomal DNA Lactococcus lactis 39
1.3 Chromosomal DNA (for enzyme reactions) 39
IV.2 BACTERIOPHAGE DNA 40
2.1. The preparation of a sterile bacteriphage stock 40
2.2 Large lysates (used for DNA isolations) 41
2.3 Continued purification of lysate’s 41
2.4 Purification and concentration of bacteriophage using CsCl density gradient centrifugation. 42
2.5 Isolating the bacteriophage DNA 42
2.6 Isoletion of bacteriophage M13 DNA template 42
IV.3 "MINIPREP" PROCEDURE FOR SMALL SCALE PLASMID ISOLATION 43
3.1 Boiling method 43
3.2 Alkaline (Birnboim) "miniprep" for B.subtilis 44
3.3 Alkaline (Birnboim) miniprep for E. coli 45
3.4 Alkaline (Birnboim) "miniprep" for L. lactis 46
3.5 Fast, one-step miniprep procedure for E. coli 46
IV.4 BIG SCALE PLASMID ISOLATIONS (B.SUBTILIS / E. COLI) 47
4.1 "Maxi prep" method 47
4.2 Alternative method (B.subtilis) 48
4.3 PEG precipitation off plasmid DNA from an CsCl solution 49
IV.5 BIG SCALE PLASMID ISOLATION L.LACTIS 49
IV.6 CALCULATING DNA CONCENTRATIONS 50
6.1 the Dische reactie 50
6.2 Absorption at 260 nm with the Spectrophotometer 51
6.3 Estimating using gel-electrophoresis 51
