TA?Cloning

TA Cloning

I. Initial mixture

5 μl dd H2O

1 μl PCR product

1 μl ligation buffer

2 μl TA vector (add second to last)

1 μl ligase (add last)

Incubate overnight at 15℃.

II. Electroporation

1. Chill cuvettes on ice before starting.

2. Set out electrocompetent JS5 cells to thaw on ice. It is important to keep them on ice.

3. Dry one 100 mg/ml AMP plate for each sample. Add 40 μl of xgal and 40 μl of IPTG to each plate.

4. Add 500 μl of SOC or SOB media to each 5 ml snap cap tube.

5. Heat ligations at 65℃ for 5 - 7 minutes, set at room temperature.

6. Dilute ligations 1:25 in water, NOT TE. This dilution is optional, 1 μl of ligation has been used effectively.

7. Add 30 μl of competent cells to each chilled cuvette.

8. Add 1 μl of ligation int℃hilled cuvettes. Use the longer tips and make sure you go all the way to the bottom of the cuvette.Tap several times to mix.

9. Turn electroporator on and hit the middle two buttons on the front at the same time. The number 1.80 should appear.

10. Put the cuvette in the electroporator and push the two pulse buttons at the same time. The lead will flash 3 times and then beep.

11. If the cuvette pops it is bad. Add electrocompetent cells to one of your extra cuvettes and try again. If it still pops, there is probably too much salt. Dilute the sample further or seek help.

12. Immediately, remove the sample from the cuvette and put into the tube with the SOC or SOB. Do not mix but flick the tube to resuspend.

13. Incubate in the shaker at 37℃ for 30 minutes to 1 hour.

14. Repeat above for each ligation.

15. After the incubation, plate 100 μl on one plate containing the xgal and IPTG.

16. Invert and incubate overnight at 37℃.

17. Check for positives by PCR

a. With internal PCR primers

b. With foward and reverse primer.

18. Pick white colonies into PCR mix, then streak on 2x YT & Amp t℃reate stock.

19. Incubate plate at 37℃ overnight.