2.2 Methods
2.2.1 Cloning and DNA Conservation
Polymerase Chain Reaction, PCR.
The amplification of DNA was carried out using the specific enzymes for each PCR according to the manufacturer’s guide. Phusion DNA polymerase (NEB, UK) was used to amplify truncated CPG2 genes and clone into pET-151-D-Topo vector (Invitrogen, UK). PCR amplification was usually carried out using a 0.5
µL DNA template (0.52 ng), 10 µL of 5◊Phusion HF Buffer, 1 µL of 10 mM of
dNTPs mix, 2.5 µL of 10 mM forward and reverse primers from table 2.3, 0.5 L
of Phusion DNA polymerase (NEB, UK) and distilled H2O to a final volume of
50µL. The reaction mixtures were briefly centrifuged at 1,500◊g, before heating in a A Biometer T3 thermocycler. The general cycling conditions used for PCR reactions involving amplification and site-directed mutagenesis are described in Table 2.4.
Table 2.4: Cycling conditions used for PCR amplification.
Cycle Step Temperature Time Cycles
Initial Denaturation 98¶C 30 s 1
Denaturation 98¶C 5 s
Annealing† 56¶C 10 s 30
Extension 72¶C 45 s
Final Extension 72¶C 10 min 1
4¶C On hold
† For PCR reactions, for primers greater than 20 nucleotides in length
the annealing step was performed at 3¶C above the lowest Tmof the
primer pair. When primers with Tm Ø72¶C were used, a two-step
thermo-cycling without a separate annealing step was used. Agarose Gel Electrosphoresis.
The results of most PCR experiments were checked by running a portion of the amplified reaction mixture in an agarose gel. The required amount of agarose (1%, w/v) was added to Tris-acetate-EDTA (TAE) buffer (Sanbrook et al. 1989) and dissolved by heating to boiling using a microwave. Safeview Nucleic Acid Stain was added (0.1%, w/v) and, once the gel was set, it was submerged in TAE buffer. DNA samples from PCR reactions were prepared by mixing with 2 µL loading dye. Electrophoresis was carried out at 50 volts for 1.5 hours or
until the dye front was at least half way through the gel. The gel image was recorded on a gel documentation system. The fragment of interest was excised from the gel under a UV trans-illuminator using a sharp blade. A QIAprep gel extraction kit (Qiagen, Germany) was used to remove the agarose from the band of interest. The DNA concentration was determined using the NanoDrop 2000c UV-Vis spectrophotometer.
Ligation of Linear DNA fragments.
PCR product bands 1 and 2 for CPG2CAT construct, composed of two DNA
fragments encoding for residues 23-214 and residues 323-415 respectively were excised and purified using a QIAquick Gel Extraction Kit after confirmation by visualisation on an agarose gel (see previous section). These two fragments were ligated at a nanogram ratio concentration of 3:1 PCR product 1: PCR product 2 in the presence of 1 U of T4 DNA ligase polymerase (Invitrogen, UK) according to manufacturer’s instruction in a volume of 20 µL. Reaction mixtures
were incubated at 16¶C overnight. The next day, the polymerase were heat inactivated at 65°C for 10 minutes before cloning in expression vector pET151- D-TOPO (Invitrogen, UK) (see next Section).
Cloning procedure.
Amplified PCR products of truncated CPG2 gene were inserted in the E.coli
expression vector pET151-D-TOPO (Invitrogen, UK). The pET Directional TOPO expression kit utilises a 5-minute cloning strategy to directionally clone a blunt-end PCR product into a vector with no ligase, post-PCR procedures or restriction enzymes required. In this system, the PCR product is directionally cloned by adding four bases to the forward primers CACC (see Table 2.3). The PCR product is stabilised in the correct orientation when the overhang in the cloning vector GTGG anneals to the added bases in the 5’ end of the PCR product. All amplified gene segments were ligated into the pET-151-D-TOPO vector with an N-terminal tag containing the V5 epitope and a hexahistidine
affinity tag, which could be removed by TEV cleavage. The cloning reaction mixture was prepared using the reagents described in Table 2.5. The mixture was mixed gently with the end of a pipette tip and incubated at room temperature (22-23¶C) for 5 minutes. For optimal results, a 2:1 molar ratio of PCR product to vector was used. Once the cloning reaction was performed, chemically competent E.coli strains were transformed with the new pET TOPO constructs (see Section 2.2.2).
Table 2.5: Cloning reaction mixture. PCR product 1µL Salt solution 1µL Sterile water 3µL Vector 1µL Total volume 6µL Site-Directed Mutagenesis.
Site-specific mutation protocol to substitute a specific amino acid in CPG2 23-415
was performed by PCR with use of the QuikChange® Site-Directed Mutagenesis Kit (Agilent Technologies, UK) and the oligonucleotides listed in Table 2.3, with the pET151-D-TOPO CPG223-415 plasmid as a template. This method allows
the production of mutants in a rapid three-step procedure involving 1) mutant strand synthesis with two synthetic oligonucleotide primers (see Table 2.3), both containing the desired mutation and each complementary to opposite strands of the vector using temperature cycling where the PCR reaction goes around the entire vector (see Section 2.2.1), 2) digestion of methylated parent plasmid
strand by using 1 µL of DPN1 restriction enzyme at 37¶C for an hour in the
PCR mixture to select for mutation-containing synthesised DNA only, and 3) transformation of 15µL of the final reaction into 100µL of DH5–competent cells.
The PCR reaction was performed using the high fidelity Phusion DNA polymerase to minimise the chances of introducing unwanted mutations in both the gene and the plasmid DNA backbone. Finally, a colony was picked, the plasmid purified and sequenced to check for correct mutation and any PCR introduced errors.
PCR Screening of Bacterial Colonies.
After ligation and plating, PCR screening was performed to test for positive insertion of the gene of interest within the vector. A well isolated colony was picked and transferred to 50 L of sterile water. Part of the colony was transferred to 10 mL LB media containing the appropriate antibiotic for overnight culture and miniprep. 1 µL of the inoculated solution was added to 9 µL of PCR pre-mix. An appropriate volume of PCR pre-mix was set up as
described in Table 2.6.
PCR was preformed according to Section 2.2.1-PCR, and the PCR products were analysed using a 1% agarose gel (see Section 2.2.1-Agarose Gel Electrosphoresis).
Table 2.6: PCR screening of bacterial colonies. 1◊Pre-Mix (µL) 20◊Pre-Mix (µL) 10x Taq buffer (+KCl, -MgCl2) 1 20 MgCl2(50 mM) 0.3 6 dNTPs 10 mM 0.1 2 Forward primer 0.1 2 Reverse primer 0.1 2 W1 detergent 0.5 10 Taq polymerase 0.1 2 distilled H2O 6.8 136 Glycerol Stocks.
The plasmid constructs produced were stored after transformation in E. coli
strains in glycerol stocks for long-term stability. 800 µL of the overnights
prepared in Section 2.2.1-PCR Screening of Bacterial Colonies were transferred to 2 mL cryogenic vials with 400 µL 50% (v/v) glycerol solution. The vials were
flash frozen in liquid nitrogen and stored at -80¶C for future use.
Purification of Plasmid DNA.
Plasmid DNA of positive clones were purified from a 5 mL cell culture in LB (see Section 2.2.1-PCR) using the QIAGEN Mini-Prep Kit (Qiagen, Germany). This kit employs the alkaline lysis method where the cells are broken open using an alkaline solution consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified by its adsorption onto a silica
matrix, which is driven by the presence of high concentrations of chaotropic salts. Manufacturers instructions were used and plasmid DNA was recovered in 30 µL
of distilled H2O and stored at -20¶C.
Sequence Analysis of Plasmid DNA.
Purified plasmid DNA was dispatched to GATC (Germany) and sequence analysis was performed using vector-specific forward and reverse T7 primers synthesised by GATC to assess the correct orientation of the insert into the vector, presence of start and stop codons and no mutations were incorporated by PCR errors.