Vanessa's Cloning Protocol

Ligation
After gel extraction and purification of digested products, set up ligation reaction as follows:

Use T4 DNA ligase and 10X T4 DNA ligase buffer

Include at least 3:1 insert:vector—can estimate based on intensity from EtBr stain.

__ml Insert

__ml Vector

£3 ml 10X T4 DNA ligase

1 ml T4 DNA ligase

£30ml Total reaction volume

Incubate at room temperature for ³2 hrs or overnight

Transform E. coli with half volume of ligation reaction

Transformation of Chemically Competent E. coli
Use DH5a cells that are already chemically competent (can get them from Invitrogen or from Shiying) stored at -80°C

1.  Place cells on ice to thaw

2.  Add DNA to cells

a.  If just transforming plasmid, use 100ng DNA

b.  If working from ligation reaction, use full or half reaction amount

3.  Incubate DNA:E. Coli on ice for 30 min

a.  Set heat block at 42°C

4.  Heat shock bacteria for 30 sec

5.  Place on ice for 2 min

6.  Add 250ml SOC media (provided with cells, or use LB media)

7.  Incubate at 37°C shaking

8.  Spin down bacterial (pulse-spin) in microcentrifuge and resuspend in 100ml SOC

9.  Plate out bacterial in LB plates with appropriate antibiotic

10. Place in 37°C incubator overnight

Pick colonies the next day and grow overnight

Analyze transformants

Crude Mini-prep to screen colonies
1.  Pellet 1ml bacteria in microcentrifuge tube.

2.  Resuspend in 150ml chilled P1 buffer.

3.  Add 150ml P2 lysis buffer, mix by inverting.

4.  Add 150ml P3 neutralization buffer, mix thoroughly by inverting until you see white precipitate.

5.  Spin down at max speed on tabletop centrifuge for 10 min at rt.

6.  Transfer 225ml to new microcentrifuge tube.

7.  Add 570µl 100% EtOH and mix thoroughly by inverting.

8.  Spin down at max speed in microcentrifuge at 4°C for 20 min.

9.  Aspirate supernatant carefully so as not to disturb pellet.

10.       Wash pellet once with 70% EtOH

11.       Dry pellet for ~5 min and resuspend in 20-50ml ddH2O.

12.       Use appropriate amount of DNA for restriction digest

13.       Run out digest on agarose gel to determine if insert is present.

14.       Grow up cultures of colonies that have insert for Qiagen spin column mini-prep.