TOPO cloning reaction allows direct insertion of PCR products into the pCR4®Blunt-‐TOPO vector, catalysed by blunt end topoisomerase. ORFs of interest were first amplified by PCR and resolved on a DNA agarose gel. The PCR product was then extracted from the gel using the gel extraction kit from QIAGEN and used for TOPO cloning reaction. TOPO cloning reactions were set up on ice as shown in Table 2.10 and were then incubated at room temperature for 15 minutes. After that, 6µl of reaction mixture was used to transform MACH1 competent cells. Cells were spread on LB agar plates containing Ampicillin (100mg/ml) or Kanamycin (10mg/ml) and were incubated overnight at 37˚C. On the next day, bacterial colony PCR (see section 2.1.2) was performed, using M13F and M13R primers, for a minimum of 4 bacterial colonies to screen for positive transformants. Mini-‐prep cultures were then set up for positive transformants and DNA was purified from bacterial culture by QIAGEN MiniPrep Kit. The purified DNA was subsequently subjected to diagnostic test digest and DNA, which was tested positive was then sent for sequencing. Glycerol stocks of bacterial colonies transformed with sequence-‐verified pCR4®Blunt-‐TOPO constructs were made as described in section 2.1.9.
Table 2.10. Reaction mixture for TOPO cloning reaction.
Reagent Volume pCR4®Blunt-‐TOPO vector 1 µl Salt solution 1 µl PCR product 4 µl Total volume 6 µl 2.1.7 Gateway cloning
Gateway cloning (Hartley et al., 2000) was performed to allow shuffling of open reading frames into plasmids of interest.
2.1.7.1 Gateway BP cloning reaction
PCR products of ORFs flanked by attB recombination sites were inserted into the pDONR223 entry vector by BP recombination reaction. BP reactions were carried out using a specific insert to entry vector ratio, which was calculated using the following formula:
Amount of insert (ng) = 125 x size of ORF x 660 106
BP reaction tubes were set up on ice as shown in Table 2.11, and incubated at 25˚C for 1.5 hour. After this, 1µl of Proteinase K (from BP reaction kit, Invitrogen) was added to the reaction mixture and incubated for 10 minutes at 37˚C to digest the BP clonase enzyme. 6µl of reaction mixture was then used to transform 50µl of XL1-‐Blue competent cells. These cells were spread on LB agar plates containing Spectinomycin (100µg/ml) and the plates were incubated overnight at 37˚C. On the next day, bacterial colony PCR (see section 2.1.2) was performed, using GateF2 and GateR1 primers, for a minimum of 5 bacterial colonies to screen for positive transformants. Miniprep cultures were then set up for positive transformants and DNA was purified from bacterial culture by QIAGEN MiniPrep Kit. The purified DNA was subsequently subjected to diagnostic test digest and DNA, which was tested positive was then sent for sequencing.
Table 2.11. Reaction mixture for BP cloning reaction.
Reagent Volume
BP clonase II enzyme mix 2 µl
pDONR223 vector (300ng/µl) 1 µl
PCR product or linearized pCR4Blunt-‐TOPO construct x µl
Sterile water y µl Total volume 10 µl
2.1.7.2 Gateway LR Cloning Reaction
Gateway LR reaction allows the shuttling of DNA insert from entry vector (pDONR223) to destination vectors by recombination reaction, catalysed by LR clonase enzyme. LR cloning reactions were set up on ice as shown in Table 2.12 and the reaction tubes were incubated at 25˚C for 1.5 hour. Following the incubation period, 1µl of Proteinase K (Invitrogen) was added to the reaction mixture and incubated at 37˚C for 10 minutes.
Table 2.12. Reaction mixture for LR cloning reaction.
Reagent Volume
BP clonase II enzyme mix 2 µl
pDONR223 vector (300ng/µl) 1 µl
PCR product or linearized pCR4Blunt-‐TOPO construct x µl
Sterile water y µl
Total volume 10 µl
6µl of reaction mixture was then used to transform 50µl of DH5α competent cells. These cells were spread on LB agar plates containing Kanamycin (10mg/ml) and the plates were incubated overnight at 37˚C. On the next day, at least 3 colonies were picked to set up miniprep cultures and DNA was purified from the bacterial culture on the following day by QIAGEN MiniPrep Kit. The purified DNA was subsequently subjected to diagnostic test digest and colonies carrying plasmids positive for test digest were used to make glycerol stocks.
2.1.8 Restriction Digest
Restriction digest reactions were set up as shown in Table 2.13 and incubated at the optimal temperature (mostly 37˚C) for the restriction endonucleases used and for 1 hour. Typically, 800ng of DNA sample was used for diagnostic test digest and 5µg of DNA sample was used for restriction cloning.
Table 2.13. Reaction mixture for restriction digest.
DNA sample x µl Restriction endonuclease 1.0 µl Reaction buffer 1.5 µl BSA 1.5 µl Sterile water y µl Total volume 15 µl 2.1.9 Restriction Cloning
Restriction cloning was performed, using the Quick Ligation kit from NEB. Insert was excised from an original vector using a pair of restriction endonucleases (see section 2.1.7), and the destination vector to which the insert is going to be ligated was also digested with a compatible pair of restriction endonucleases (i.e. the sticky ends generated following restriction digest are complementary to each other). Typically, 100ng of restriction-‐digested insert was incubated with vector at a molar ratio of 1:3. Ligation reactions were set up as shown in Table 2.14 on ice, and were incubated at room temperature for 10 minutes. 6µl of reaction mixture was then used to transform 50µl of DH5α competent cells. These cells were spread on LB agar plates containing Kanamycin and the plates were incubated overnight at 37˚C. On the next day, at least 4 colonies were picked to set up miniprep cultures and DNA was purified from the bacterial culture on the following day by QIAGEN MiniPrep Kit. The purified DNA was subsequently subjected to diagnostic test digest and colonies carrying plasmids positive for test digest were used to make glycerol stocks.
Table 2.14. Reaction mixture for ligation reaction.
Restriction digested insert (100ng) x µl
Restriction digested vector y µl
2X Quick Ligase Buffer 5 µl
Quick Ligase 0.5 µl
Sterile water z µl
Total volume 10 µl