Blunt End Ligation

PCR

1, Run PCR using Pfu or Phusion DNA Polymerase. Do not use Taq DNA polymerase

2, Run PCR product on agarose gel.

3. Cut out desired band, add gel solubilization buffer QG.

Vector Digestion

4. Digest pET30b:

5 µl of pET30b
0.5 µl of EcoRV
0.5 µl of CIP
1.5 µl of 10X NEB Buffer 3
1.5 µl of 10X BSA
water to 15 µl.
37 oC for 1 hour.

5. Run gel and cut out linearilzed vector.

6. Add some QG buffer, dissolve gel with QG buffer, mix with solution from step 3.

7. Purify DNA using spin column.

8. Elute DNA using 20 µl water.

9. Add 2.5 µl 10X ligase buffer.

10. Add 0.5 µl T4 ligase.

11. Ligate at room temperature for 2 hours.

12. Transform 100 µl Top10 competent cells with 10 µl ligation mix.

Ligase buffer:

Contains 5% PEG

10x Ligation buffer (containing 1 ul of 10 mM ATP) 2.5 µl T4 DNA ligase 0.5 µl 30% PEG 8000 4 µl 10X Ligase buffer: 0.5 M Tris pH 7.8, 0.1 M MgCl2,  50 mM DTT, 2 mM EDTA,  4 mM rATP, 5% PEG 8000

Vector: add 1 µl Biolabs calf intestine alkaline phosphatase. Incubate 37° for 1 hr.