CHAPTER 2: Materials and Methods 42
2.2 Molecular Genetic Techniques 45
2.2.1
Plasmid DNA extractions from Escherichia coli using commercial kits
Overnight cultures (5 ml and 100 ml) of transformed E. coli in Lysogeny broth (LB) broth with the appropriate antibiotic for selection were pelleted in preparation for mini-‐preps and midi-‐ preps respectively. Small-‐scale isolations were carried out using GeneJETTM Plasmid Mini-‐prep
Kit (Fermentas) according to the protocol provided. Plasmid DNA was eluted in 50 μl distilled H2O. Medium scale isolations were performed according to the instructions supplied with the
QIAfilterTM Plasmid Midi Kit (Qiagen) and DNA was re-‐suspended in 200 μl dH2O. A list of
plasmids/replicons used during this project is outlined in table 2.1 at the end of this chapter.
2.2.2
Virus RNA extractions from cell culture supernatant
RNeasy Mini Kit (Qiagen) was used for RNA extraction from supernatant recovered from virus infections or transfections according to the manufacturer’s instructions. RNA was stored at -‐80°C and was thawed in an ice bath when required.
2.2.3
PCR product column clean up
QIAquickPCR® Purification Kit (Qiagen) was used for purification of DNA from PCR reactions
according to the manufacturer’s instructions. DNA was stored at -‐20°C until required.
2.2.4
Extraction of DNA from agarose gel
Deoxyribonucleic acid fragments were extracted from agarose gel using the DNA Extraction Kit (Qiagen) according to the manufacturer’s instructions. The DNA was eluted in 20 -‐ 40 μl dH2O
and quantitated by spectrophotometry and then used for all down-‐stream activities.
2.2.5
Transformation of E. coli with plasmid DNA
Around 3 μl ligation mixture (or 1 μl plasmid DNA where appropriate) was added to 50 μl ice-‐ thawed α-‐Select Chemically Competent Escherichia coli (Bioline) and stored in an ice bath for 20 mins. The mix was placed in a 42°C waterbath for 35 seconds before being returned to ice for 2 mins. Five hundred microlitres of SOC medium (Sambrook and Russell, 2001) was added before
incubating the tube in a 37°C shaker (225 rpm) for 1 hour. One hundred and seventy-‐five microlitres of cells were plated onto LB agar plates supplemented with appropriate antibiotic selection and incubated overnight at 37°C.
2.2.6
Ligation of DNA fragments
Digested DNA fragments or PCR products were ligated into vectors at a molar ratio of 3:1 (insert:vector) in a 20 μl reaction containing 1 x Ligation Buffer (400 mM Tris-‐ HCl, 100 mM MgCl2, 100 mM dithiothreitol (DTT), 5 mM adenosine trisphosphate (ATP) (pH 7.8 at 25°C)) and
5 U T4 DNA Ligase (Fermentas). Reactions were incubated at room temperature for 2 hrs and used directly without further purification for bacterial transformations.
2.2.7
DNA sequencing
All sequencing reactions were set up in a 10 μl total volume mix using 25 pmol of appropriate primer. Plasmid sequencing required 400-‐500 ng, whereas PCR fragment sequencing required 100-‐400 ng. GATC Biotech sequenced all DNA samples.
2.2.8
Restriction enzyme digestion
Digestion of DNA was carried out using the manufacturer’s recommended amount of restriction enzyme in a solution containing 1 x the specific supplied reaction buffer, and if required, with 100 μg/ml bovine serum albumin (BSA). Incubation temperature and subsequent thermal inactivation were carried out according to the manufacturer’s instructions. The digestion reaction was incubated from 1 to 4 hrs before being run ona 1% weight per volume total (w/v) agarose gel for size/banding pattern analysis. The restriction sites used to linearise plasmids during this project are outlined in table 2.2 at the end of this chapter.
2.2.9
In vitro reverse transcription (cDNA synthesis)
Reverse transcription reactions were carried out using Superscript II Reverse Transcriptase (Invitrogen). Ten microlitres of purified RNA (at unknown concentration) was incubated in a mixture containing 100 pmol oligo dT (Invitrogen), 10 mM dNTP mix and 1.5 μl H2O for 5 min at
65°C. Following a 2 min cool on an ice bath, 0.2 μmoles DTT, 1 x Superscript Buffer (250 mM Tris-‐HCl, pH 8.3 at RT, 375 mM KCl, 15 mM MgCl2), and 20 U RiboLock RNase Inhibitor
(Fermentas) was added and incubated for 2 mins at 42°C. Two hundred units of Superscript II were added to the reaction before a final 50 min incubation at 42°C and reaction termination at 70°C for 15 mins. The cDNA mixture was stored at -‐20°C while not in use.
2.2.10
Amplification of DNA fragments (up to 3kb) -‐ PCR
A master mix was prepared containing 1 x Taq Buffer with (NH4)2SO4 (750 mM Tris-‐HCl (pH 8.8
at 25.5°C), 200 mM (NH4)2SO4, 0.1% Tween-‐20), 2.5 mM each deoxyribonucelotide triphosphate
(dNTP), 30 pmoles of the relevant forward and reverse oligonucleotide, and 2.5 U Taq DNA Polymerase (Fermentas) in a 50 μl reaction volume.
Thermal cycling comprised 30 -‐ 40 cycles as follows: one cycle of denaturation for 2 mins at 95°C, 29-‐34 cycles of denaturation for 1 min at 95°C, annealing for 30 sec at 55°C (or different according to oligonucleotide conditions) and elongation for 1 min/kilo base pairs (kps) at 72°C, and a last cycle of 5 min at 72°C. A list of oligonucleotides is recorded in section 2.9 at the end of this chapter.
2.2.11 Site-‐directed mutagenesis
Point mutations were introduced into cDNAs using QuikChange® II XL Site-‐directed
Mutagenesis Kit (Stratagene). Briefly, 20 to 40-‐mer oligonucleotides were designed for opposite strands of the plasmid and incorporated nucleotide mutations necessary for the project. The plasmids were amplified by PCR using the mutagenic oligonucleotides in table 2.3 according to the provided protocol. Competent E. coli cells provided with the kit were transformed with amplified plasmid and individual clones sequenced to confirm the presence of specifically introduced mutations. The mutated regions were subsequently sub-‐cloned into a reliable, characterised construct to ensure no additional off target changes were incorporated and sequenced to confirm.
2.2.12 Overlap extension PCR
This method was used when multiple changes to virus cDNA was required. Polymerase chain reaction oligonucleotides that were complementary to the virus cDNA and carrying the desired mutation(s) were designed to ensure that mismatches were at least 10 base pairs (bps) from
either the 5’ or 3’ end of the oligonucleotide. Two DNA fragments that overlapped in the region based upon the mutagenic oligonucleotides were produced using appropriate external
oligonucleotides. Oligonucleotides used to construct pRLucΔCRE_3’CRE and pPV3-‐LIKE are shown in tables 2.4 and 2.5 respectively. Following column purification, the two DNA fragments were then used in a linear polymerase chain reaction that lacked the external oligonucleotides. Briefly, 9 cycles of PCR consisted of:
95°C -‐ 2 minutes 95°C -‐ 30 seconds 56°C -‐ 30 seconds
72°C -‐ Variable depending on length of product (typically 1 min per Kb) Followed by a final extension:
72°C -‐ 5 minutes 4°C -‐ Hold
The two external oligonucleotides were then added at standard concentrations as
recommended by the manufacturers protocol. Twenty-‐nine cycles of PCR were then used at standard conditions, using annealing temperatures best suited for the two external
oligonucleotides being used. Extension time was also based upon the size of the desired product.
2.2.13 Construction of pSL3Δ, ΔpT7Rep3-‐L and ΔpRLucWT
pT7Rep3-‐L and pRLucWT were digested with PmlI and PacI and re-‐ligated following Klenow fragment treatment to produce suitable blunt ends. This effectively removed the entire IRES and the majority of the luciferase coding region. Similarly, pT7FLC/SL3 was digested with XhoI and SalI and re-‐ligated, removing the polymerase-‐coding region. No Klenow fragment treatment was required given the complementarity in sticky-‐ends.
2.2.14 In vitro transcription
Linearised plasmid for RNA transcription was first prepared by column purification using the QIAquickPCR® Purification Kit (Qiagen) and re-‐suspended in RNase-‐free water. T7 MEGAscript
Between 1.5 and 2 μL of RNA was confirmed on a 1% agarose gel before column purification using RNeasy Mini Kit (Qiagen) and quantification on a spectrophotometer.