Colony PCR with Eukaryotic Cells? (View forum version)



cell the truth

Posted 17 February 2010 - 02:52 AM

Hi Everybody

I try to find an 850bp Insertion into the gDNA of HEK cells. Therefore, we like to do this by PCR, but we don't like to isolate DNA and the whole procedure. The PCR product has not to be really clean or usable for cloning or whatever, it just should bring up a band (or not) on a Et-Br stained gel. I. e. we are looking for a "quick'n'dirty" method. From my work with bacteria, i know that there it is possible to do it as Colony PCR. Is this possible with eukaryotic cells? Any suggestions?

Thanks for advice and help

bob1

Posted 17 February 2010 - 03:08 PM

it should work, though eukaryotes have a lot more DNA per cell than prokaryotes so there may be problems with releasing all the DNA into the PCR. Also, bacteria fit nicely onto the end of a tip, whereas eukaryote cells are much larger, so will be harder to pick.

gebirgsziege

Posted 17 February 2010 - 11:26 PM

the size does not matter here; eukaryotic cells are still so small that you will pick bilions of them with a single tothpick.....besides you have special toothpicks with a tip-diameter of a frew Ám.

I used sucessful quick and dirty preps for hyphomycetes and yeasts....just put into a ball mill, boiled them in TE-buffer for 10 min and used this as template for sequencing. but fungi have a very strong cell wall which needs the prior treatments.....I think you should give it a try using a thermostable enzyme incubating your cells a 98░ for like 5min before PCR conditions....but I think I got some ads about "direct PCR" from tissue cultures, cannot remember the company but you should be able to google it.
Nevertheless you can have inhibiting substances in your prep which require DNA extraction.

cell the truth

Posted 19 February 2010 - 06:42 AM

Thanx, we'll try. Enjoy ur weekend.

bob1

Posted 21 February 2010 - 03:43 PM

the size does not matter here; eukaryotic cells are still so small that you will pick bilions of them with a single tothpick.....besides you have special toothpicks with a tip-diameter of a frew Ám.

In my experience with human cells trypsinised for picking clones, they will go up a 10 ul pipette tip only 3 at a time maximum which isn't much. Having said that there should still be plenty of DNA for a PCR.