T7 ligation

1. Make ligation mix:

For ligation of PCR product or insert from another plasmid:

10 ul 2X ligation buffer, 10 ul water.

For ligation of annealed oligo mix from another plasmid:

10 ul 2X ligation buffer, 10 ul annealed oligo mix

2. Mix gel slice from vector and insert.

3. Add QG buffer to dissolve gel, incubate at 60 oC for a few minutes until dissloved.

4. Load solution to spin column. Spin 15 sec.

5. Wash column  2 X  with 0.35 ml PE buffer.

6. Spin 1 min at max speed.

7. Add 20 ul ligation mix to column

8. Spin to a clean tube.

9. Add 0.5 ul T7 ligase.

10. Ligate for 1 hour at 25 oC (room temperature).

11. Take 10 ul out to transform 100 ul competent cells.

12. Spin down cells and spread cells on LB plate (with selection antibiotics).

To make annealed oligo  mix:

1. take 5 ul 10 uM of Top (sense) oligo, 5 ul of 10 uM of Bottom (antisense) oligo. Mix, add 1 ul of 10X Taq PCR buffer.

2. Heat at 95 oC for 2 to 5 min, using heat lid.

3. Turn off PCR machine, let machine cool down slowly to room temperature.

4. Spin briefly if there is any water on the lid.