Modified, from Kayoko Kimbara, Ginsberg Lab UCSD, August 2004
Updated Sept 2006
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:
1. Determine the concentration of your plasmid stock
Using the Spectrometer to calculate the concentration of your DNA:
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
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.)