Hello
I extracted Chlamydomonas genomic DNA. and i checked gel electrophoresis.
But i can't find genomic DNA in gel. and it remained smear.
i didn't treated RNase.
Can you check my protocol?
1) add 500uL TEN buffer.
2) resuspend, spin 10sec. and aspirate off supernatant.
3) resuspend cells in DW on ice and add 300uL of SDS-EB buffer, voltex.
4) Extract once with 350uL phenol/CIA(1:1) for few min by vortex, separate phases by centrifugation for 5min. transfer aq. to a new tube.
5) Extract once with 300uL CIA(24:1), centrifugation, transfer aq. to a new tube.
6) Add 2 volumes abs.ethanol, incubate on ice for 30min, centrifuge for 10min, wash pellet once with 200uL 70% ethanol.
7) Dry pellet and resuspend in 30uL DW.
what is problem in my experiment??
may i know the detailed composition of TEN and SDS-EB buffer. i think you got a good quantity of DNA, but the problem is with its quality.
It appears as if the DNA had already been sheared by the action of nucleases (DNase). The problem may be with the solution used during the resuspension of the DNA template. You also may need to add proteinase K in order to digest the nucleases.
Hi Dear Jun-Woo Lee
I think, in your electrophoresis, smear density is very high. You once repeat the test again with smaller amounts of DNA samples and with high concentrations of agarose gel.
Best, Roshdi
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