Transformation

Gel Purification of vector and insert

1. After digestion, run DNA on an 0.8~1% gel, 0.5 X TAE, 250 V for ~ 10 min.

2. Cut out DNA band.

3. Add ~ 3 vol of QG buffer.

4. Incubate at 60 oC until gel are completely dissolved.

5. Transfer to a silica column

6. Spin for 15 sec

7. Wash column with 2 X of 0.35 ml PE, spin 15 sec

8. Spin 1 min

9. Add 25 ul ddH2O, use new sterilzed collection tube (1.5 ml microcentrifuge tube)

10. Spin for 1 min, collect flow through (purified DNA). Sometimes it is necessary to check if there is any DNA by agarose gel

Ligation

1. Add 2.5 ul 10 X ligation bufer, 1 ul of T4 ligase to the purified DNA.

2. Incubate at room temperature for 30 min to  2 hours, or overnight.

The ligation product can be stored at -20 oC for a few weeks.

Transformation

1. Put ligation product on ice.

2. Take 1 vial of Top10 or 10G or NEB Turbo or DH5alpha or NEB5alpha 

(for plasmid without LTR) or STBL3 or NEB Stable (for lentivirus or adenovirus construct).

3. If the vial with competent cells has > 100 ul, while it is still in solid state, use pipet tip to dig out the ice (~100 ul),

put it into ligation mix (entire ligation mix).

Optionally, Add 1 μl of DMSO or PEG8000 (40%) to cells + ligation mix.

4. Mix gently, incubate on ice for 15 to 30 min, usally 20 min

5. Heat shock at 42 oC on a PCR machine for  30 seconds upto 1.5 min

6. Put on ice for 1 to 5 min

7. Add 500 ul  SOC or other recovery medium (LB)

8. Incubate at 37 oC  with  shaking at 750 rmp (eppendorf shaker) or  180 rpm (shaker incubator) for 30 to 2 hours as below:

Recovery time: 

30 min for amp-R plasmid,

1 ~ 2 hour  for Kan-R plasmids,  some plasmid with Kan resistance must incubate for 2 hours.

9. Spin down bacteria for 30 sec.

10. Dump off liquid, leave about 50 to 100 ul.

11. Resuspend bacteria with pipette.

12. Transfer suspension to LB plate with antibiotics.

13. Spread using beads.

14. Incubate at 37 oC oven overnight. Make sure the agar side is up.