MS 5 5 2017

Subcloning:

1.prepare plasmid

2. 15ul per tube

3.10X Fast Digestion Buffer 1.5ul/tube (4oC)

4.Nor I and KPN I restricted digestion, 0.3ul/tube

5.spin to get rid of bubble.

6.5ul mix/tube. If the concentration is high, add 5ul H2O and 5ul plasmid. If the concentration is low, add 10 ul plasmid. Make sure 15ul/tube.

7.spin to get rid of bubble.

8.37oC incubation, 30~60 min.

9.add 5ul GRGreen loading Buffer.

10.Use 5ul Fluorescent DNA Ladder II, EG-258 as ladder.

11.Run agarouse gel, 150V

12.cut down the strip of interest, add 1ml QG buffer, disolve in 90oC water.

13.transfer liquid into mini column, spin at 13,200 rpm for 1 min.

14.wash with 350ul PE buffer, twice. then spin again.

15.wash with 20ul ddH2O.

16.add 2.5ul 10X T4 LB

17.add T4 ligase 1ul.

18.incubate in RT, 1h

19.transfer into 100ul NEB Turbo, incubate on ice for 15min.

20.Heat shock at 50oC, 2min.

21.add SOC 500ul, culture at 37oC for 30min.

22.spin down cell, drop off supernatant, resuspend and transfer to LB/Amp solid media.