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rmbio

Member Since 26 Jan 2009
Offline Last Active Apr 16 2013 03:51 AM

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In Topic: No amplification during RT-PCR

16 March 2013 - 08:54 AM

Thanks for reply jerryshelly1.

It was less than a week old. If RNA is a problem, shouldn't amplification of other genes also get hampered? Yes, with previous cDNA current primers are giving brilliant amplification. No, I don't expect downregulation of the gene in the present cDNA. With the same set of PCR reagents previous cDNA is amplifying; also amplifying is the other set of genes with the present cDNA. So I don't blame the PCR reagents.

Thanks

In Topic: cDNA amplification problem

22 February 2013 - 03:14 AM

I have experienced the same problem, where I used to get the amplification with the taq polymerase, but with pfu polymerase the gel used to be blank. Try using Advantage DNA polymerase mix from clontech. I found it really useful giving the opposite results. It gave the amplification with the primers which did not work with the normal taq pol.

In Topic: ChIP sonication

13 December 2012 - 04:47 AM

I did not do any experiment after above attempt.
What's the marker size in your case? I have consistently observed that if u load the sonicated samples directly (without phenol purifyling and having 1% SDS coming from lysis buffer in my case) onto the agarose gel, you get some weared pattern. I am sure you have done the same. So my suggesiton is that after RNase-proteinaseK incubation give phenol-chloroform treatment followed by alcohol (isopropanol or ethanol) precipitation and than load the samples on gel for analyzing the size.

In Topic: O/E site

21 November 2012 - 11:11 PM

thanks!!

In Topic: Phenol wash necessary?

10 July 2012 - 02:43 AM

I ran a gel meanwhile and it gave goo bands at my expected sizes. I thus came to the conclusion that there is no effect of RNase and proteinase K on the agarose gel performance of the DNA

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rmbio

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