Mutagenesis by PCR
Join two PCR fragments by PCR
[edit]use 945 as example
1. First PCR two fragments
Fragment 1
945-pMBP-Xba1-F atacTCTAGA ACACAGGAAACAGCCAGTCCGT
947-6H-R GATTACCAGTTTACCTTCTTCgtgatggtgatggtgatgGATTTTGGCGAGAGCCGAGGC
Fragment 2
947-6H-F GCCTCGGCTCTCGCCAAAATCcatcaccatcaccatcacGAAGAAGGTAAACTGGTAATC
945-pMBP-E3C-Nco1-R (AATAACAATAACAACAACCTCgaagttctgttccag ggt ccgCATATGTCCATGGatctc)
gagatCCATGGACATATGcggaccctggaacagaacttcGAGGTTGTTGTTATTGTTATT
using 1281 pMAL-p5E or 1282 pMAL-p5E-AvPAL as template
2. Run PCR product on agarose gel
3. Cut out desired PCR fragments from gel, put into one 1.5 ml microcentrifuge tube
4. Add 1 ml QG solution.
5. Purify DNA using miniprep column, elute with 20 ul H2O
6. PCR again using
945-pMBP-Xba1-F
atacTCTAGA ACACAGGAAACAGCCAGTCCGT
945-pMBP-E3C-Nco1-R
(AATAACAATAACAACAACCTCgaagttctgttccag ggt ccgCATATGTCCATGGatctc)
gagatCCATGGACATATGcggaccctggaacagaacttcGAGGTTGTTGTTATTGTTATT
template is 1 ul of purified PCR products from step 5.
7. Purify PCR fragment as step #2 to #5.
8. digest purified fragment with Xba1 +Nco1
9. Digest vector pT7A30 with Xba1 +Nco1
10. Purify digested vector on gel as step #2 to #4
11. Add digested PCR from step #8 to the tube on step #10.
12. Purify on miniprep column
13. Elute with 20 ul H2O.
13. Add 2.5 ul of 10X T4 DNA ligase Buffer
14. Add 0.5~ 1ul T4 DNA ligase
15. Ligate at RT for 30 min
16. Transform 10G competent cells.
17. Spread on LB/Amp plate.
18. Grow at 37 oC o/n
19. Pick colonies and grow on 5 ml 2xYT/amp o/n
20. Make miniprep and digest with Xba1+ Nco1
21. Send positive clone out for sequencing.