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PCR directly from eukaryotic cell without DNA extraction
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PCR directly from eukaryotic cell without DNA extraction
It is known that PCR could be done directly on bacteria cells. However, is
it any equivalent protocol for that performed on eukaryotic cells? Please
direct me to any resource about that. ... ...
PCR directly form the cells
18.06.2003 03:10
293-T cells; 10cm2 dish;
took 10ul of cell culture (out of 2ml trypsinized cells); 100 C in PCR machine for 5 minutes (it breaks cell, kills most of the proteins). on that set up 100 ul PCR reaction. works perfectly fine. nice and clean product. good luck ..
PCR directly from eukaryotic cell without DNA extraction
14.06.2003 07:37
in my test, 10 or more spore of fungi is not good as 1-5 spores, may be protein inhibit the reaction; as to 1 spore, normal 40 circles have no enough amplicon for electrophoresis,
so i take nested pcr, everything is OK, why don't you try more circles, 60-70circles may be good,
Last update 28-Nov-2000, Rating Good of 0 votes.
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i think the quantity of cell is important. if too much ,the protein should inhibit the pcr,too little shall supply few template.and the fore-prepare of cell is necessary!
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I am doing multiplex PCR directly from agar plates by resuspending the one single colony in 100ul sterile MilliQ water, vortexing and leaving it to stand for at least 10 min. Finally, i add 10ul of the bacterial suspension to 40ul master mix. It has worked for me. But then, you have to consider the type of bacteria you are handling. mine are staphs.
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I doubt it will work if you are not joking. But I am wondering why we can directly pick bacterial clone to amplify without any pretreatment. It's hard to imagine one can expose genomic DNA from bacterial and eukaryotic cells as PCR template without extra steps to break membrane and get rid of proteins.
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Easy, just pick a couple of cells and boiled in ddH2O. Take a few of microliters to be template. Either way, use pestal to break
the cells and dilute in water. Take a few ul to be template.
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