Editing Cloning of PCR Product

== Cloning of PCR product by Restriction Digestion and Ligation  ==

1. Run PCR for 25 cycles using 1 unit of Phusion 

2. While doing PCR, digest vector with 0.5 ul of NEB restriction enzymes using NEB buffer. 

2. Run gel, cut out desired bands; do not run vector digestion on gel at this step. 

3. Add QG buffer to dissolve the gel slice at 50 oC. 

4. Purify DNA fragment using spin column. Wash two times with 350 ul PE. Do not wash with PB. Spin additional 1 min to get rid of residual enthanol after PE washes. Elute DNA with 20 ul water. Do not discard the column. Label it and put it on a rack. 

5. Add 2.5 ul of NEB buffer, 0.5 to 1 ul NEB enzymes, digest for 2 to 4 hours. 

6. Run gel to check if the fragment is good (no smearing). Cut out DNA ban. Also run gel to purify digested vector 

7. Put vector and PCR gel slices to one tube. 

8. Add QG buffer to dissolve gel slices, load to the spin column previously used at step 4. Wash two times with 350 ul PE. Do not wash with PB. Spin additional 1 min to get rid of residual enthanol after PE washes. Elute with 20 ul water. 

9. Add 2.5 ul 10 X T4 ligase buffer, 0.5 ul T4 ligase, ligate at room temperature for 30 min to 2 hours. 

Alternatively, elute DNA using 15 ul water (step 8), add 2X or 4X Fast Ligation Buffer (to 1X final). Add 0.5 or 1 ul Fast T4 ligase, ligate for 5 min. 

10. Transform 100 ul competent cells with the entire ligation mixture. Make sure it is heat shock for 1 min to 1.5 min if you are not using thin-wall tube. 

11. Cool on ice for 1 min. 

12. Add 350 ul SOC, incubate at 37 oC shaking for 15 min to 1 hour. 

13. Spin down cells, leave about 100 ul liquid. 

14. Use pipette to resuspend cells. Transfer all cells to LB plate. 

15. Add 4 ~8 beads, swirl for 30 seconds. 

16. Pour off beads to a collection bottle/flasks, do not discard beads. 

17. Place plate agar side up in a 37 oC incubator. Incubate at 37 oC o/n. 

18. Scree colonies using one gene-specific primer and one vector specific primer.<br> 

[[Category:Protocols]]