September 5, 2013
- QuickChange Site Directed Mutagenesis of V0200 to remove BSMBI cut site in the Hygro resistance sequence
- Use 50 ng of dsDNA template (BD002) and 125 ng of each primer.
- Template strands are about 7kb for BD002.
- Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μL concentration.
- Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μL concentration.
- DNA template (BD002) miniprep concentration: 88ng/μL.
| Reagents || BD002
| Plasmid DNA (50 ng) || 0.6 μL
| primer 1 (10 μM, 125 ng) || 2.7
| primer 2 (10 μM, 125 ng) || 2.66
| 10x reaction buffer|| 5.0
| dNTP mix || 1.0
| dH2O || 38.04
| Total || 50.0
- No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme.
- Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to the sample reaction
- Thermal cycling
- 95°C/ 30 sec
- [95°C/ 30 sec; 55°C/ 1 min; 68°C/ 7 min (1 min/kb plasmid length)]x18
- 4°C, ∞
- DpnI Digest (gets rid of methylated template DNA)
- Add 1 μL DpnI enzyme to the reaction.
- Gently and thoroughly mix the reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA
- Transform 10μL of Dpn I treated DNA ftom the reaction tube to the 50μL aliquot of XL1-Blue supercompetent cells (Comes with the QuikChange Kit) No colony grow
- Transform 40μL of Dpn I-treated DNA from the reaction tube to the 50μL aliquot of DH5α-T. One colony grow
- Transform 1μL of control plasmid from the kit to 50μL of XL1-Blue supercompetent cells. 100s colonies grow
- Do the traditional transformation process: 10minutes on ice, 45 seconds at 42°C, 1 minute on ice, add SOC and incubate 45 minutes at 37°C, Spin down the cells and resuspend the pallet with 100μL LB AMP broth, spread on AMP agar plate and incubate for 18 hours.