Sub clon in g Procedures

2.1.3.1. Restriction Endonuclease Digestions

Restriction enzymes were obtained from Boehringer M annheim, New England Biolabs, USB, or Promega, and used according to the m anufacturer's recom m endations. All reactions were allowed to proceed to completion, unless otherwise stated.

2.1.3.2. Fill-in Reactions

The 3' recessed ends of digested DNA fragments were filled in with Klenow fragment of DNA polymerase I (Boehringer Mannheim) or AMV-reverse transcriptase (Life Sciences Inc.), to create blunt DNA termini. Reactions were performed under buffer conditions recom m ended by the manufacturers. Klenow (2 units/50pl) reactions were allowed to proceed for 5 minutes at room temperature, and reverse transcriptase (20 units/50|il) reactions for 30 minutes at 37°C.

2.1.3.3. Dephosphorylation of DNA Termini

W here appropriate, to prevent the circularization of the vector fragments with blunt termini during the incubation with DNA ligase, the phosphates from DNA termini were removed with calf intestinal alkaline phosphatase (GIF; Boehringer Mannheim). Dephosphorylation reactions were allowed to take place in volumes of 50|il, in the following buffer conditions: 50mM Tris (pHS.O), lOOpM EDTA. The total incubation at 37°C was 1 hour, and the GIF

78

enzym e w as added at two points: one unit in the beginning of an incubation and one unit after the first 30 m inutes. R eactions were term inated by phenol extraction, and phenol- extracted DNAs precipitated with ethanol.

2.1 .3 .4 . P h o s p h o r y la t io n o f D N A T erm ini

T he term ini o f double-stranded DNA oligonucleotide inserts w ere phosphorylated to enhance their ligation efficiency. The single stranded components of oligonucleotide probes used in SouthW esternfw ere phosphorylated in the presence of 40pCi [y^^PjATP/pg DNA in order to label them radioactively. Both phosphorylation reactions were perform ed by using T4 polynucleotide kinase (B oehringer M annheim ). The phosphorylation buffer

co n tain ed 50m M T ris (pH 8.2), lOmM MgCl], lOOpM ED T A , 5mM D TT, lOOpM

sperm idine.

2 .1 .3 .5 . M u n g B ean N u cle a se R ea ctio n s

To rem ove both 3' and 5' single-stranded extensions from DNA term ini to leave ligatable blunt ends, m ung bean nuclease (N EB iolabs) was used. The reaction buffer'contained 30m M sodium acetate (pH4.6), 5()mM NaCl, Im M ZnC l2. DN As w ere digested with 50 units o f M BN (Pharm acia) for 5 m inutes at 16^C, after which the reactions were stopped by adding Tris (pH9.0) to lOmM, LiCl to 0.5M and SDS to 1%. R eactions w ere then extracted with phenol and precipitated with ethanol.

2 .1 .3 .6 . B a l3 1 E x o n u c le a s e R e a c tio n s

Bal31 nuclease degrades both 5' and 3' termini of duplex DNA without generating internal scissions, and was used^for progressive shortening of double-stranded DNA. The reaction buffer containedf6(X)mM N aCl, 12mM C a C li, 12mM M gC l], 20m M T ris (pH8.0) and Im M EDTA. Reactions (40pl) were terminated by adding 60pl volume of T E(10:l)/20m M EG TA, w hich specifically chelates the essential cofactor Ca^+, together with lOOpl phenol (pH8.0) and 20pg tRNA. Phenol extracted samples were then precipitated with ethanol.

2 .1 .3 .7 . Iso la tio n o f D N A F r a g m en ts

N ativ e p o ly ac ry la m id e gels (6-15% ) w ere used to purify ra d io a ctiv ely labelled oligonucleotides and closely migrating fragm ents less than 500bp; the electrophoresis was performed in vertical gel boxes, with TBE (90mM Tris-borate/2mM EDTA) as both gel and running buffers. DNA w as located by ethidium bromide staining and U V -illum ination, or

79

by autoradiography (if fragments were radioactively labelled). DNA fragments were eluted from gel slices in 400^.1 o f the elution buffer (IM ammonium acetate, 1% SDS and ImM EDTA) at 3 7 ^ overnight

DNA fragments larger than 500bp were separated by agarose gel electrophoresis and eluted from LM P agarose (EMC Bioproducts) by adding at least five volumes of TE (20:1; pHS.O), heating the mixture at 7(PC for 10 minutes, and phenol extracting the samples.

2.1.3.8. DNA Ligation Reactions

T4 DNA ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA, and was used to join restriction fragments together. The reaction conditions were: 66mM Tris (pH7.5), 5mM MgC12, ImM DTT, Im M ATP, and 0.1-0.5 units/pl T4 DNA ligase (Boehringer M annheim). Where applicable, the vector:insert-ratio was varied between 1:3 and 1:5. Ligation reactions were allowed to take place at the optimal temperature 16°C for 4-16 hours.

For ligation reactions involving blunt DNA termini, the reaction conditions were modified, so that the final ATP concentration was 50pM. This is believed to enhance the ligation efficiency of blunt termini.

2.1.3.9. DNA Transformation into Competent

Strains

Cells of the E. coli strain SCS I (F‘ , en d A l, gyrA96, thi-1, hsdR 17 [nc-mk+], supE44, recA l, relA l, X') (Stratagene) were made competent by the method of Hanahan (1985). A single colony of the SC S-1 strain was used to inoculate an overnight culture of 2ml in L- broth. This was used to further inoculate 400ml L-broth, which was split into four 1 liter sterile flasks. The cultures were grow n^^vigorous shaking at 37°C until their optical density (A.=550nm) was within the range 0.45-0.55. The cultures were cooled on ice, and pelleted by a centrifugation at 4°C with minimal force. Thoroughly drained bacterial pellets were suspended in 120ml of cold R Fl (lOOmM RbCl, 45mM M nC li, 35mM potassium acetate, lOmM C aC li, 5mM M gCli, 0.5mM LiCl, 15%(w/v) sucrose; pH5.8) and left on ice for 15 minutes. Cells were pelleted and drained as above, after which they were resuspended in 30ml of cold RF2 (lOmM MOPS, lOmM RbCl, 75mM CaC12, 15%(w/v) glycerol; pH 6.8) and left on ice for 15 minutes. Aliquots o f this suspension were dispensed into Eppendorf tubes and quickly frozen in liquid nitrogen. By this method, transformation competences exceeding lO^cfu/pg DNA were routinely achieved. An 100- 200|il aliquot of competent bacteria were used for each transformation event.

80

To transform com petent bacteria a ligation mixture or pure plasmid DNA was added to them, and left on ice for 20 minutes. The transformants were then heat shocked in a 45°C waterbath for 2 minutes. After this 1ml of L-broth was added and transformants incubated at 37®C for one hour. The transformation mixtures were finally plated out on LB-agar (1.5%) plates containing l(X)pg/ml ampicillin.