We want to use PCR on small cell numbers and avoid loss of DNA by DNA purification procedures.
Therese,
We use Viagen Mouse Tail reagent for single C elegans PCR (~1000 cells) which works fantasically. The amount we put in the PCR probably is about 5 cells worth of the lysis. The also have a "cell" version too (as opposed to mouse tail, although they would probably work just as well).
http://www.viagenbiotech.com/index.php/directpcr-lysis-reagent-mouse-tail.html
Cheers,
Steve
If you want to start from single cell then any mild detergent at low conc is enough. We use Triton X-100 0.2% (2 ul) and perform the RT in 10 ul. The following PCR is then done in 25 ul w/o purification. However, conc of Tween-20 or Triton around 1% are ok as well as guanidine-HCl at low conc (about 50 mM in RT). Guanidine is good if you want to break the nucleus, the other detergents show variability from cell to cell, but in most cases you will get only cytoplasmic RNA from the lysis.
You can try the PCR with cell just add 1% SDS along with it and Initial denaturation for 20 minutes. Hope the PCR end product will give good result
'HotSHOT' DNA method is quite simple to perform with the use of alkaline lysis reagent and neutralisation buffer- all can be made in the lab with the common chemicals. It is mainly used on tissue samples but I have tried this with cell samples and works fine on PCR.
Look at below for more information
http://www.med.umich.edu/tamc/HotShotDNA.PDF
No need for a special kit. Just lyse the cells in an appropriate buffer and protK (maybe after freeze/thaw). Heat deactivate the protK and then do a normal PCR with the crude extract. Works for worms (they have a thick cuticle), thus should work for cells.
the best one from sigma as follow:
CelLytic™ B Cell Lysis Reagent For bacterial cell lysis, standard strength (http://www.sigmaaldrich.com/catalog/product/sigma/b7435?lang=en®ion=IQ)
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