Colony PCR with Eukaryotic Cells?
Posted 17 February 2010 - 02:52 AM
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
Posted 17 February 2010 - 03:08 PM
Posted 17 February 2010 - 11:26 PM
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.
Posted 19 February 2010 - 06:42 AM
Posted 21 February 2010 - 03:43 PM
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.
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.