Nucleic acid manipulation

2.2.4.1 Polymerase Chain Reaction (PCR)

PCR reactions were carried out in 50 µl volumes containing 2.5 µl of each primer (10 µM), 5 µl 10X PCR reaction buffer, 5 µl dNTP mix (2 mM each), 3.5 µl MgSO4

solution (25 mM), I U of KOD Hot Start DNA polymerase (Merck) and 1 µl of template DNA (1-10 ng/µl). Reaction conditions were - initial denaturation of 5 min at 95°C followed by 30 cycles of denaturing at 95°C for 30 seconds, primer annealing at 45 – 60°C for 30 seconds, extension at 70 °C and final extension of 70 °C for 5 min. Samples were then held at 4°C. Primer annealing temperature was determined by primer Tm and extension time was determined by the length of the amplicon, using an amplification speed of KOD polymerase set at 25 seconds per 1 Kb of amplicon size.

Quick-change and excision PCRs

For introducing specific mutations into a plasmid a quick-change (site-directed mutagenesis) PCR was carried out. Here, complementary 30 – 35 nucleotide primers were designed targeting the region where the change was to be made. The specific mutation to be introduced was designed in the centre of the primer sequence.

Excision PCR was used to delete specific nucleotides. Here, 18 – 25 nucleotides long primers flanking each side of the deletion were designed in outward directions.

The PCR cycle number was reduced to 18 cycles. PCR reactions were then treated with 1 U DpnI and incubated for 1 h at 37°C. 1 in 10 dilution of this reaction in nuclease-free water was then used to transform competent JM109 bacteria.

Reverse transcription polymerase chain reaction (RT-PCR)

2 ng/µl RNA with 1.5 µl of RT primer (10 µM/ml) was added to DNase/RNase-free water making up a total reaction of 25 µl. The reaction was then heated to 74°C for 5 min before rapidly cooling on ice for 5 min. To this 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega), 10 µl 5X buffer, 1 µl 10 mM 4xdNTPs and 40 U (1 µl) of RNaseOUT (Life Technologies) were added and the reaction

incubated at 42°C for 2 h. Samples were then heated to 90°C for 10 min to inactivate the enzyme before being stored at -20°C until use in a PCR reaction. PCRs were performed using 5 µl of the synthesised cDNA in a 50 µl reaction containing 2.5 µl of each primer (10 µM/ml), 5 µl 10X PCR reaction buffer, 5 µl dNTP mix (2 mM each), 3.5 µl MgSO4 solution (25 mM) and I U of KOD Hot Start DNA polymerase (Merck).

PCR conditions were followed as described above.

2.2.4.2 Cloning

Restriction digests were performed in a reaction volume of 50 µl containing 1 U of restriction enzyme per 1 µg of DNA and 1X final concentration of appropriate enzyme buffer. Nuclease-free water was used to make up final reaction volume. Reactions were incubated at 37 °C for between 1 to 4 h. Digested DNA was then analysed using agarose electrophoresis gels and extracted and purified using a gel extraction kit (Promega). Ligation

Prior to ligation the vector was linearised either using PCR or restriction digestion methodology. Samples were then dephosphorylated with CIAP (calf intestinal alkaline phosphatase; Promega) by addition of CIAP Reaction buffer and 0.1 U CIAP per µl (final volume). Reactions were incubated at 37 °C for 1 h before being purified using a gel purification kit (Promega). Ligations were performed using T4 DNA ligase (Promega) overnight at RT, using a 1:3 and 1:5 molar ratio of vector to insert. The amount of insert DNA to be added was calculated using the equation below.

ngofvectorXsizeofinsertinbp

sizeofvectorinbp Xmolarratioof

insert

vector =ngofinsert

bp = total number of base pairs. Infusion reaction

Infusion cloning was used as an alternative to restriction enzyme based cloning. Plasmids were linearised using restriction enzymes or by excision PCR. Excision PCR primers for linearising the plasmid (vector) were designed to incorporate the terminal 15

nt of the insert. Primers for the insert sequence were then designed to include 15 nts of the linearised plasmid boundaries. PCR products thus contain 15 nt complementary sequences at each end. 100 ng of insert, 50 ng of vector and 2 µl 5X InFusion enzyme pre-mix (InFusion HD Cloning Kit; Clonetech Laboratories Inc.) along with nuclease- free water made up to a volume of 10 µl was incubated at 50°C for 15 min. The reaction mix was then placed on ice for 5 min before using the entire 10 µl to transform competent JM109 bacteria. Bacterial cells were plated on LB-agar with appropriate antibiotic, and incubated at 37°C.

Colony PCR

Bacterial colonies were screened for insert using colony PCR. Individual colonies were resuspended in 13.5 µl of DNase free water and heat shocked at 95 °C for 5 min before cooling on ice. PCR was carried out using 4 µl 5X PCR buffer, 0.4 µl dNTP mix (10 mM), 1 µl forward primer (10 mM), 1µl reverse primer (10 mM) and 0.1 µl GoTaq polymerase (Promega). PCR conditions were - initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 seconds, primer anneal for 30 seconds, extension at 72°C final extension at 72°C for 10 min; followed by cooling and storage at 4°C. Bacterial colonies that were positive for the insert were then used to inoculate 5 ml LB- broth cultures for DNA preparation using Qiagen QIAprep Spin Miniprep kit (Qiagen, Germany). Plasmids were then sequenced via Sanger sequencing (Source BioScience). Agarose gel electrophoresis

DNA fragments were visualised using agarose gel electrophoresis. Agarose gels were prepared by melting agarose (Bioline) in 1X TBE buffer (89 mM Tris-Borate, 2 mM EDTA, pH 8.3; Sigma-Aldrich) or 1X TAE buffer (40 mM Tris-acetate and 1 mM EDTA, pH 8.3; Sigma-Aldrich) with ethidium bromide (10 mg/ml, Biotechnology grade; AMRESCO). The solution was microwaved in order to melt the agarose and left to cool at RT. The solution was then poured into a horizontal gel chamber with a comb and left at RT until set. DNA samples were prepared by adding an appropriate amount of Gel Loading Dye (in a ratio of 1:6 of dye to sample). A maximum sample volume of 50 µl was then loaded onto the agarose gel. DNA bands were separated at 75 – 100V for at least 30 min in running buffer. DNA fragments were then visualized using a UV

transferred into eppendorfs and then purified from the gel using Wizard® SV Gel and PCR Clean-Up System (Promega), according to manufacturer’s recommendations. If required, DNA bands were sequenced via Sanger sequencing (Source BioScience). Preparation and maintenance of plasmid stocks

JM109 cells were made chemically competent using the Mix & Go E. coli

Transformation Kit & Buffer Set (Zymo Research).

All plasmids used in this project contained Ampicillin selection genes and so transformation of these plasmids in competent cells involved only 5 mins on ice. 5 ml LB-broth cultures (starter culture) were inoculated with the transformation mix for 18 h at 37°C with constant agitation. For large-scale plasmid preparation, 150 ml cultures were inoculated with 1.5 ml of the starter culture for another 18 h. Cultures were centrifuged at 4,000 rpm for 30 min at 4°C. Supernatant was discarded and the bacterial pellet re-suspended in re-suspension buffer for either small-scale DNA preparation (Qiagen QIAprep Spin Miniprep Kit; Qiagen; Germany) or large-scale DNA preparation (NucleoBond Xtra midiprep kit; Macherey-Nagel; Germany). Protocols for DNA extraction were according to manufacturers recommendations. DNA concentration was determined on a NanoDrop spectrophotometer (Thermo Scientific). DNA preparations were stored at -20°C.

Cultures containing cDNA plasmids for viral M-segments or the glycoprotein were grown at RT with constant agitation for 36 h.

Generation of bacterial glycerol stocks

500 µl from bacterial cultures containing sequence-validated plasmids were mixed with 500 µl of sterile 50% glycerol. Glycerol stocks generated were stored at -80°C for long- term storage.

The sequences of all plasmids generated in this study have been confirmed by Sanger sequencing (Source BioScience).