quick pcr (colony screening by PCR)

john hess jfhess at ucdavis.edu
Fri Mar 18 17:22:31 EST 1994


OK, here's another way;

08:30, you come into the lab in the morning, check the incubator, you've
got colonies!!!

Put 10ul of 2X YT/amp in a 500 ul tube.

Using a yellow tip, carefully pick a colony from the plate and swirl it
around in the 500 ul tube with YT/amp in it.

Set up PCR reactions for final volume 25 ul. Penultimate addition is 1 ul
of bugs from tube with 10 ul of media in it. Last addition is mineral oil.

While the PCR machine is running, use 5ul of bugs to innoculate 2 mls of
LB/amp for each sample. Number the tubes the same as the PCR tubes!

PCR for 20 cycles, add 10 ul of glycerol dye to the PCR tube, tap the
tube hard to mix dye with aqueous phase. Stab pipet tip thru oil to
remove sample for gel. No need to use new tubes, or change tips, this is
the screen only!!!!!!

Once you know which PCR reactions are positive, 1) toss the miniprep
cultures that are negative, 2) use the remaining 2-3 ul of bugs to start
a 250 ml culture for large scale plasmid prep.  3) let the 2ml cultures
that are positives grow until 4 pm, then crank out alkaline lysis/KAc
minipreps.  Resuspend DNA in 50 ul of RNase TE, use 10 for OH-
denaturation for sequencing, etoh ppt, let air dry O/N. 
out of lab at 5:30


next day, clean plates, pour gel.
While you sequence and run the gel, harvest the 250 ml culture and do
plasmid prep.

I have done this for pSP72 clones and (minus sequencing) for pT7-7
expression clones. I always put DNA into pSP72 for probes/sequencing
general, then put into expression vectors for protein production.
So far so good.

John Hess, PhD                    Phone me 916 752 8420
Dept of Human Anatomy             FAX me 916 752 8520
University of Calif               Email me jfhess at ucdavis.edu
Davis, CA                         or leave me alone, your choice.



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