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Problem with Semiquantitative PCR

Semiquantitative

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#1 Rifat Rumki

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Posted 28 September 2011 - 09:35 AM

Hello everyone. I'm an MS student and currently doing my thesis research on Molecular Biology and the organism I'm studying on is Jute. Currently I'm facing a problem with semiquantitative PCR. I'm working on a stress-responsive gene in Jute, which I identified using degenerate primers. After sequencing, I analyzed the gene using Genscan and found the gene of 2.6 KB size. The predicted coding sequence was 906 bp. Now when I performed RT-PCR and trying to analyze the expression pattern of the gene by semiquantitative PCR, its amplifying ~2.8 KB product from cDNA. I'm using normal conditions of PCR. It seems to be impossible to amplify such a big product. The bands are sharp and single band is seen. Can anyone help me Please with solving this problem?????? Let me know for further queries if necessary. I've attached the picture of the gel of PCR products.

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#2 Kamran

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Posted 29 September 2011 - 12:50 AM

~2.8Kb band is indication of genomic DNA amplification.

I'd suggest you to treat your RNA prep with DNase to get rid of any contaminant DNA before proceeding further.

Best of Luck!

Kamran

#3 Rifat Rumki

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Posted 29 September 2011 - 06:42 AM

Thank U Very Much Kamran for Ur kind suggestion..... Hope U've checked my gel photo.... Usually genomic DNA contamination (If present) shows bands above The highest marker of the Ladder. And I'm sure about the primers which does not work on the genomic DNA..... I did PCR with genomic DNA and the primers I've used for semiquantitative PCR and result was negative (no amplification occurred). So definitely it is amplifying using cDNA as template. It is very unusual event since it requires especial conditions and reagents to amplify such huge product from cDNA. Please, I would be very happy if U have any further suggestion for me.

#4 Adrian K

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Posted 29 September 2011 - 07:45 AM

IF this is the case I suggest you to clone and sequence your ~2.8kb fragment to find out...
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#5 Rifat Rumki

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Posted 29 September 2011 - 09:01 PM

Thank U Very Much Adrian.... I also thought about it.... But it will be a very large fragment to be cloned since the vector we use in our lab can accommodate maximum 2.0 KB fragments ..... However, I prepared this sample for sequencing..... Hope to get something out of it...... Actually now I'm a bit worried about my primers..... Those primers showed match with Arabidopsis RING/U-box domain containing protein (Helicase/Ubiquitin) in BLAST result. Database showed me an mRNA/cds sequence of the helicase protein of 2.75 KB..... So unless I get the sequence I cannot say whether it is amplifying the gene of my interest or it is helicase..... Posted Image

Thank U Once again for ur kind suggestion..... Please share with me if U've any new idea..... Posted Image




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