Paul.xu: Created page with "1, Prepare Gibson Mix, aliquot 5 ul to tubes. 2, Run PCR using High Fidelity Polymerase such as KOD, Phusion, Q5, 20 ul reaction. 3, Digest vector..."

2020-06-12T13:01:28Z

Created page with "1, Prepare Gibson Mix, aliquot 5 ul to tubes. 2, Run PCR using High Fidelity Polymerase such as KOD, Phusion, Q5, 20 ul reaction. 3, Digest vector..."

New page

1, Prepare [[Gibson_Assembly|Gibson Mix,]] aliquot 5 ul to  tubes.

2, Run PCR using High Fidelity Polymerase such as KOD, Phusion, Q5, 20 ul reaction.

3, Digest vector using a pair of restriction enzymes

4, Gel purify PCR product and vector, cut out both vector and PCR product bands

5. Put in a tube, add 3X QG buffer , dissolve in hot water bath

6. Cool down to RT, transfer to a spin column

7. Spin for 15 sec, dump liquid

8, Wash column 2 X with 300 ul PE buffer, dump liquid

9, Spin again at max speed for 1 min

10, Elute DNA using 5 ul water

11, Add 5 ul Gibson mix, mix

12, Incubate at 50 oC for 30 min,  store at -20 oC  or do transformation immediately

13. Transform NEBTurbo, 
[[Category:Protocols]]

Cloning PCR Product by Gibson Mix - Revision history

Paul.xu: Created page with "1, Prepare Gibson Mix, aliquot 5 ul to tubes. 2, Run PCR using High Fidelity Polymerase such as KOD, Phusion, Q5, 20 ul reaction. 3, Digest vector..."