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PCR on cDNA and SGamplification???


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#1 clange

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Posted 29 February 2012 - 05:50 AM

I have noticed throughout my amplifications, single genome or bulk amplifications, that whenever I don't get a PCR product from my reaction, I can see a band in the well of my gels. This has been occurring regardless of what polymerase kit I use or using fresh water and Tris for my dilutions and volumes, so I don't think it is contamination from any of the components of my MMXs or diluents. So far the only explanation I have come across for seeing bands in the wells is it being amplification of genomic DNA, but my samples come from serum, not cells, so the chances of this seems really low.

I've attached a pic of my most recently failed attempt at SGamplifications and one that worked in the past but the sequences from the amplicons seen were not clean.

I ran a bulk amplification reaction in parallel with the most recent SGamplifications. The bulk amplification has worked in the past, under the same conditions and with the same reagents and concentrations. But this time, it also did not give me a result and I can see this band in my well.

I use the Qiagen RNA extraction kit to get the RNA from the virus Invitrogen Superscript III for cDNA synthesis. My RNA and cDNA yields respectively after each step for this particular sample are 63.6ng/ul and 60ng/ul (by Nanodrop)

IMG0082A.jpg
Above are failed PCRS

IMG0084A.jpg
Above are 2 supposed single genome amplicons that gave dirty sequences after analysis. You can also see the bnds in the wells of the negative reactions (which I think is the source of contamination wrt my sequences)

My PCR conditions for both 1st round and nested PCRs

PCR MMX
Phusion HIFI buffer 5ul
dNTPs (10mM) 0.5ul
primer 1 (50uM) 0.25ul
primer 2 (50uM) 0.25ul
Phusion Taq pol 0.5ul
dH2O 17.5ul
template 1ul

denaturation
94oC for 3 mins
35cycles
94oC for 30secs
optimized annealing temp for 30 secs
72oC for 4 mins
final extension
72oC for 10 mins

#2 Trof

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Posted 06 March 2012 - 10:07 AM

I'm not familiar with the term SGamplification. So you are doing RT-PCR on virus RNA from serum. Do you treat with DNAase?
That thing in wells look like genomic DNA yes. But I find unlikely that this high-weight DNA would come from amplification (what is the size of your longest band in marker?). You ca try to make a reaction and run it on gel without amplification to see if it was there beforehand. Anyway it looks like there is so much of it in reaction, other thing you can try is to lower the amount of template like 10x or 100x.

Other option is that it's not long DNA at all, but something having a big difficulty passing through gel and binding EtBr. Maybe some protein-DNA complex - wild guess.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

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