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I wish to amplify a 2.5 kb gene from RNA. My colleague had isolated RNA, synthesized cDNA and I had amplified the gene using this cDNA previously. When I did RNA isolation and cDNA synthesis using the same RTase, I am unable to get any amplification for the same gene with all the PCR reagents same as used earlier. About 6 other different PCRs with the amplicon size of less than 200 bp (for qPCR) are working well with equal efficiency in both the cases. The starting tissue for both the case is almost the same, so I don't expect that this gene is not expressed in the tissue aliquote which I am using.
Is the larger amplicon size a problem and shall I expect that because of some problems during my RT reaction, larger RNAs have not completely reverse transcribed? Are there any modifications which can be made during cDNA synthesis to get rid of this problem?
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ChIP sonication
03 October 2012 - 05:21 AM
Hi
I have been trying to optimize sonication of the chromatin from rat brain for ChIP. Please find below the gel image of the decrosslinked, proteinase K treated and phenol washed DNA. Sonication was done with Bioruptor (1) 5 (2) 10 (3) 15 and (4) 20 cycles of 30 sec on/off at high power. Does any of these chromatin samples appears to be of the quality of being taken ahead for immunoprecipitation? I can provide details of steps performed after tissue harvest and before sonication.
Thank you in anticipation
I have been trying to optimize sonication of the chromatin from rat brain for ChIP. Please find below the gel image of the decrosslinked, proteinase K treated and phenol washed DNA. Sonication was done with Bioruptor (1) 5 (2) 10 (3) 15 and (4) 20 cycles of 30 sec on/off at high power. Does any of these chromatin samples appears to be of the quality of being taken ahead for immunoprecipitation? I can provide details of steps performed after tissue harvest and before sonication.
Thank you in anticipation
