advices - (Jan/17/2006 )
Hi! I just started to do molecular biology and I would need some good advices. :)
I have got some bacterial pellet and I should isolate the genomic DNA. Does anyone know a good protocol?
Then I shoul clone a gene of which I know the sequence. Can I do directly the PCR on the entire genomic DNA using the primers specific for the gene?
Can I even do the PCR using directly the bacterial pellet?
what kind of bacteria? you can do a rough lysis protcol with your bacteria involving boiling the resuspended pellet, but if you are using a bacteria with a heavy-duty cell wall you may need to proceed further into lysis. and, if it doesn't work, you have to grow up another culture to repeat the PCR; you can't really keep the lysed cell-template around without degradation...that is really the only downside compared to a full-on chromosomal prep
you should be able to use gene-specific primers on genomic DNA to get your gene of interest, that is how it is done 
you should be able to use gene-specific primers on genomic DNA to get your gene of interest, that is how it is done
Thanks! Methanococcus and they grow at pressures of up to more than 200 atm and at an optimum temperature of 85 degrees C. It is a strict anaerobe, and, as the name implies, it produces methane. In my lab I cannot grow up an other culture
but I have enough pellet. Could you perhaps tell me the lysis protocol?
I don't know if I can help you; I do not know much about the characteristics of the cell wall in methanococcus
I work on staphylococcus and you need to resuspend the pellet and do some incubations with lysostaphin, SDS, and proteinaseK; then you boil it for 10 or 15 min to obtain PCR template
for e coli (to screen clones), I resuspend the pellet and then just boil it
I use TEN for resuspension (TE, NaCl, EDTA, from Maniatis)
but your conditions may be different based on cell wall composition
Might be very well that you have a real tough cell wall there, at least from the growing conditions it sounds like that. So it is probably useful to look into RNA-preparation protocols from Mycobacteria here in the forums as those discuss the lysis of the problematic cell wall too.