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pcr problem

pcr problem - PCR - Polymerase Chain Reaction Forum

pcr problem - 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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  #11  
Old 12-19-2009, 02:10 AM
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Default Re: pcr problem

hi everyone,

thank you guys for all the great information. Aga..i already take note for everything you told for the last post.Thanks..i'll try to do the checklist again for those sample that i havent get any result..

selvam..all the best for you, thanks for the sharing..good for you that you solve your problem.

here come the other problem guyshihihi.. after the PCR, and we load into the gel for electroforesis, i got band, but that band is not good enough (means it just a tiny, smear band) and below of the band got smear which is so light...the smear is more light rather than the band...ohh i hope you guys understand what i'm sayinghihihi..

for the information here some of my volume(ul) of the reagent i'm using

MgCl 3, buffer 5, dNTP 0.5, primers(2) 4each, taq 0.125, ddh20 8.175, DNA 0.2

I'm doing PCR-RFLP for IGS region..

thanks guys
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  #12  
Old 12-19-2009, 08:23 PM
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Default Re: pcr problem

Are these electrophoretic results just after PCR or after RFLP? Cause you said you're doing PCR-RFLP and I got confused.

If it's after PCR then it seems like there is some additional product although I don't know what are the sizes of this and your desired product.
Have you tried to reduce magnesium concentration? High Mg conc. may cause more unspecific products (but in some protocols small amounts just don't work well).
Because your desired product is faint you probably have problems with reaction efficiency or PCR inhibition. Or if DNA fragment you amplify is complicated for the polymerase (long, rich in CG bases) you may also get low amount of product after PCR. In that case change polymerase to Pfu for example.
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  #13  
Old 01-30-2013, 04:39 AM
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Default Re: pcr problem

[Only registered users see links. ] It's your first day as Dr Adlard's research assistant. You are to follow his instructions to run a PCR experiment so that you can easily detect QX disease in Sydney Rock Oysters. The problem is that you have jumbled some of his instructions. Now you have to sort out the mess! On the bench in the lab are Dr Adlard's essentials for PCR, primers and polymerases. Read the labels carefully before you start.
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Old 02-14-2013, 07:09 PM
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Default Re: pcr problem

hy

nearly, i face the same problem then i'll explain it.
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