A Dohlman lab Protocol

by Ginger Hoffman

1. Titer library.

a) make the following dilutions of Library DNA: (Keep all dilutions on ice)

10-5
10-6
10-7
10-8
10-10

b) Transform by electroporation.

  • for each transformation, use 20 ul electro-competent DH10B cells thawed on ice
  • and 1 µl DNA mixed GENTLY with pipette.
  • for electroporation, use 25 µF at 200 ohms and 2.0 kV
  • make sure time constant is around 4 seconds
  • (This is using a Bio-Rad Gene Pulser System)
  • IMMEDIATELY after electroporation add 500 µl SOC to cuvette, pipette up and down a few times to mix cells. Transfer to a larger tube containing another 500 ul of SOC Shake at 37oC for 1 hour.

c) Plate transformation in order to titer.

  • transfer cells shaken at 37oC to a sterile microfuge tube.
  • spin for 1 minute at 1/2 speed.
  • aspirate supernatent with glass pipette and add 100 µl SOC.
  • plate on LB + amp and incubate at 37oC overnight.
  • next day, count colonies on each plate and determine which DNA dilution gives the desired number of colonies. When performing the actual transformation, use TEN TIMES this amount of DNA.

2.Prepare for the transformation

a) Mix 750 ml of 2x LB + 0.4% ULTRA LOW GELLING TEMPERATURE AGAROSE (SeaPrep brand from FMC)

autoclave

b) take LB + agarose out of autoclave immediately after it’s finished and place in 37oC water bath.

  • allow LB + agarose to cool to 55oC and add 30 ug/ml of Ampicillin (225 µl of 100 mg/ml stock in 750 ml LB + agarose).
  • put 25 ml of LB + agarose + amp in a 50 ml falcon tube. Repeat for 20 separate tubes. Keep tubes at 37oC until cells are added.

3.Transform Library

a) While autoclaving LB + agarose, Complete 5 separate transformations…

  • for each, use
    • 20 µl electro-competent DH10B cells
    • 1 µl DNA (at 10-fold higher concentration than
    • needed for desired number of colonies)
    • 25 µF; 200 ohms; 2.0 kV
  • put in 1 ml SOC (in 15 ml pop-cap tube) and shake at 37 oC for 1 hour.

4.Grow Library.

a) To each 25 ml Falcon tube containing LB-Amp-Agarose add about 230 µl of cells/SOC (one transformation will be distributed over 4 tubes).

b) Put tubes in an ice water bath for about 45 minutes (to let agarose solidify).

c) CAREFULLY transfer to warm room (37oC).

d) Let grow two nights. You should see little colonies suspended in the LB + agarose. Often colonies will sink to bottom of tube, this is ok.

5.Midi-Prep Colonies (Qiagen Midi-Preps).

a) Put the contents of two 50 ml falcon tubes into one centrifuge tube.

b) Spin for 20 minutes at 8000 rpm at 4oC.

c) Remove supernatent, add 1/2 the volume of the tube of dH2O.

d) Spin 15 minutes at 8000 rpm.

e) Remove supernatent, resuspend each tube in 2 ml P1 (Qiagen Midi- Preps).

f) Follow rest of Qiagen instructions for Midi-preps, resuspend final product in TE8. Pellet may be difficult or impossible to see. This is ok.

NOTES: Efficiency of amplification will depend of size of library vector. This protocol was written for LEU2 CEN library (vector is about 10.5 kb – including insert) If amplification doesn’t work, it would be useful to try changing the DNA concentration, the agarose concentration, and/or the amp concentration.