PCR trouble shooting, help, suggestions
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PCR trouble shooting, help, suggestions
If your PCR amplification somehow performs unexpectedly, it is usually caused by one of the listed possible errors - ranked by frequency.
You may try checking if the problem is repeatable, see trouble shooting flowchart.
Be patient and follow a standard scheme when setting up your reactions.
Avoid short-cuts, and be sure to ... ...
URL: PCR trouble shooting, help, suggestions
Content: original article
Rating: *****
Last update 05-Jun-2001, Rating Good of 2 votes.
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i designed one primer whr i want to amply 650 bp region in dna from human blood for my project i.e prion protein polymorphism in humans. but am not at all getting amplification nd tried more than 25 rxns. modified in different ways. wht could be the problem.
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I am running the RAPD PCR not amphlified i dont no the problem please suggest any changes. 25 microlitter reaction perkinelmer thermal cycler used
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sir iwant to amplify 4.5kb fragment by using Pfu turbo enzyme but i am not able to do this my primers NTPs enzyme all is right suggest me some thing please
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If you are amplifying a PCR product (which is a transgene) from untransformed tobacco I'd consider a few things. Firstly, could you have contaminated (very slightly) any of the PCR reagents/water etc...I would start by changing all of these including the Taq enzyme (don't throw them away, just use new until your sure they're fine). Use filter tips for this reaction at least during problem diagnosis. Finally, could it be that the primers have a target in the untransformed tobacco DNA, or that the transgenic tobacco is giving a single-primer amplification product? If you have transformed with say, a closely related Solanales gene from tomato, then you might well expect PCR primers aimed at that gene to amplify the same gene in tobacco. Alternatively, clone and sequence the product. Another lastly! I always try to do DNA extractions is the order untransformed, then transformed, so that the pestle and mortar I grind the non-transformed tissue contains no transgene. Of course, this isn't a problem with a Southern since the cross-contamination will not show up (no amplification of cross-contamination)
Hope that helps someone!
Jon
Webmaster for http://MolBiol.Net
Portal for Free Online Bioinformatics Tools
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I designed one primer pair to amplify a 135 bp region coding for N-terminal of protein but apart from getting desired product,also I am getting some non-specific amplifacation
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I am getting amplification of same size in a sample which should not give. I have tried temp. upto 68 degree. Amplification is faint as compared to the positive control.
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I am getting amplification of same size in a sample (Untransformed genomic DNA of tobacco)which should not give any amplification. I have tried temp. upto 68 degree. Amplification is faint as compared to the positive control.Please advice.
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