Editing Mutagenesis by PCR

== Join two PCR fragments by PCR ==

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&nbsp;&nbsp;1282 pMAL-p5E-AvPAL&nbsp;as template&nbsp;

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&nbsp;using miniprep column, elute with 20 ul H2O

6.&nbsp;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&nbsp;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&nbsp;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.
[[Category:Protocols]]