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
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#1
cell the truth
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Posted 17 February 2010 - 02:52 AM
If you are not part of the solution you are part of the precipitate.
#2
bob1
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Thelymitra pulchella
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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.
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gebirgsziege
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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 µm.
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.
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.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)
#4
cell the truth
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Posted 19 February 2010 - 06:42 AM
Thanx, we'll try. Enjoy ur weekend.
If you are not part of the solution you are part of the precipitate.
#5
bob1
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Posted 21 February 2010 - 03:43 PM
gebirgsziege, on Feb 17 2010, 11:26 PM, said:
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 µm.
