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Problems with PCR-no bands

Problems with PCR-no bands - PCR - Polymerase Chain Reaction Forum

Problems with PCR-no bands - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 06-30-2010, 01:07 PM
Pipette Filler
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Default Problems with PCR-no bands



Hiya,

I am new to this forum and also new to PCR, I am a student at uni studying molecular parasitology.
I have only done PCR once before and I was handed a working protocol and genomic DNA and all reagents. No thought required!

This time however I have been flung in the deep end with my project and I have very little help.

I am trying to amplify the BoLA DRB3 gene. I have extracted DNA from lung tissue and when I spec it the conc. is approx 2000ng/ul for all of my samples (200).

I made the following master mix for my PCR (based on previous PCRs)

14ul H2O
2ul 10x buffer
0.5ul each primer (100ng/ul)
2ul dNTPs (10mM)
1ul MgCl2 (25mM)
0.2ul Taq

1ul DNA (used varying concentrations after realising that the 1st time my conc was probably too high, used 2000ng/ul then I tried again with a 1:50,1:100 and 1:200 dilution)

Total volume was 20ul (19ul master mix and 1ul DNA)

PCR program was as follows:

94 2 mins, 94 30 sec-57 30 sec-72 1 min (30 cycles), 72 3 mins.

the TM of my primers are: forward 63.3 and reverse 62.

Size of fragment expected 281bp

Any help gratefully recieved! Sorry if its too long but have read other problems and people always seem to ask for more info!

Thanks,

Sarah
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Old 06-30-2010, 03:01 PM
Pipette Filler
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Default Re: Problems with PCR-no bands

Do you now have genomic DNA or cDNA?

If it's cDNA then I'd say you've too much template. I would dilute it 1 in 1000 as you want about 1-2ng cDNA in a PCR. Too much template will result in no band.

If it's genomic DNA then it's possible your primers are designed from the cDNA and spans an intron so it can't bind to your genomic DNA.

I would also add 1ul of your dNTPs as too high a concentration will inhibit your reaction. I would use twice the amount of taq just to be sure as well. Finally, you're adding 0.5ul of 10ng/ul primer, whereas I usually add 2uL of primer at a concentration of 10uM. If you find the information that came with your primers you can work out the molar concentration of your primers and adjust them to this concentration. I usually work at 50uL final volume too.

Hope this helps!
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Old 07-01-2010, 07:50 AM
Pipette Filler
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Default Re: Problems with PCR-no bands

Hello,

Thanks.

Its genomic DNA I am using and the primers I am using have been used in two previous papers also with genomic DNA.

I finally managed to find someone in the lab to help me (my supervisor is not lab based and I have just been given lab space in another lab so no one is that keen to help me as "im not their student!") and they recommnded I changed my primer and dNTP concentration the same as you have.

I will try it now and fingers crossed.

Thank you very much!

Sarah
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