Linearizing Gene-Targeting (Knockout or Knockin) Constructs for Electroporation into ES Cells

Modified, from Kayoko Kimbara, Ginsberg Lab UCSD, August 2004
Updated Sept 2006

Isolate enough targeting vector/plasmid

To get about 150ug of construct (plenty)

  1. Culture 250mL of bacteria (LB/Amp or other selective drug)
  2. Purify plasmid DNA by
    • Promega Wizard+ Maxi (Cat# A7270)
    • or Qiagen Maxi Kit (cat# 12162)

To get about 400ug of construct (excessive)

  1. Culture One Liter of bacteria (LB/Amp or other selective drug)
  2. Purify plasmid DNA by
    • Promega Wizard+ Mega (Cat #A7300)
    • or Qiagen Mega Kit (cat# 12181)

The final size of the plasmid is often over 20-kb, and to get a good yield from the Qiagen prep one needs to modify the P1 solution before use as follows:

  • add glucose (final amount in P1 solution 1% i.e. 1 gram/100mls)
  • add lysozyme (final amount in P1 solution 5mg/ml)

Procedure

1. Determine the concentration of your plasmid stock

Using the Spectrometer to calculate the concentration of your DNA:

  1. Set spec to 260
  2. Use a quartz cuvette, and get baseline standard of water alone
  3. Create a 1 to 50 dilution of your plasmid in water
    Usually these cuvettes hold 100ul, so 2uL plasmid in 98uL water
  4. Run your sample. The reading should be between 0.200 and 0.500 to be linear for calculations. You may need to run another sample that is more or less dilute to get to within this range.

Now, calculate the concentration:

Dilution factor x OD260 x 50 = DNA concentration (ug/mL)
(...50 is a constant for the calculation. It has nothing to do with the fact that I suggested a 1 to 50 dilution above.)

For example, if you did a 1 to 50 dilution and got a reading of 0.256, then:

50 x 0.256 x 50 = 640ug/mL

If your stock DNA in the eppendorf tube is in 100uL (one-tenth of a mL), then you have about 64ug in the tube. If your stock DNA is in 50uL (one-twentieth of a mL), then you have about 128ug in the tube.

2. Digest 150ug plasmid

Place 50ug/tube: total 3 tubes. You must use tubes that are OK with Phenol/Chloroform. Some tubes will melt with these solvents

The KI ES cell Facility needs 80-100ug, so starting with 150ug in 3 tubes will be fine.

50 ug construct DNA
30 ul appropriate Buffer
30 ul 10xBSA
5 ul Enzyme (NEB:20 units/ul)
+ water --- Total 300 ul/tube

Mix well and incubate them for 4hrs-ON at 37°C

3. Test 2ul on an agarose gel to make sure that the digestion is completed

4. Purify the linearized DNA

  1. Add 300ul Phenol/Chloroform(isoamyl alcohol ok) to each tube and shake gently
  2. Spin tubes on max speed for 15-30 sec.
  3. Carefully transfer the upper (aqueous) phase to a fresh tube
  4. Repeat steps a-c (Start using a cell culture hood)
  5. Add an equal volume of chloroform (no phenol) and repeat step b and c
  6. Estimate the volume of the aqueous DNA solution and add 1/10th volume 3M sodium acetate (pH 5.2). This gives a final concentration of ~0.25M NaOAc. Mix well.
  7. Add 2 volumes of ice-cold 100% ethanol. Mix well (no vortexing) and chill to -20°C for 1hr-ON
  8. Centrifuge at 4°C for 15min
  9. Wash DNA pellet with 1ml 70-80% ethanol twice.

5. Give DNA in 1ml 70-80% ethanol (per tube) to the ES Cell Facility, labeling with construct name and the approximate ug of DNA in each tube (40ug if you started with 50ug). Do not forget other required items (map, picture of gel showing linearization, etc.)