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PCR Failure..pls help!


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#1 phaffia

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Posted 05 December 2004 - 08:19 PM

Hi all,

I have been trying to clone a 2.4kb fragment at the 3'end of a viral genome but to no avail. Have tried re-isolating the RNA from the virus and re-generated my cDNA...along with newly designed primers. Nothing seemed to work. I can churn out short fragments of about 80 bp at different regions but not the large fragment.

I have also tried using polymerase systems like BD Advantage 2, DyNAzyme Ext, Platinum Taq...and different RT-PCR systems like Promega Imprompt II, Thermoscript & Superscript III.

Is there anything else that I should have tried?

Please help. Thanks in advance.

phaf

#2 netnus

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Posted 06 December 2004 - 07:22 AM

Hi all,

I have been trying to clone a 2.4kb fragment at the 3'end of a viral genome but to no avail.  Have tried re-isolating the RNA from the virus and re-generated my cDNA...along with newly designed primers. Nothing seemed to work. I can churn out short fragments of about 80 bp at different regions but not the large fragment.

I have also tried using polymerase systems like BD Advantage 2, DyNAzyme Ext, Platinum Taq...and different RT-PCR systems like Promega Imprompt II, Thermoscript & Superscript III.

Is there anything else that I should have tried?

Please help. Thanks in advance.

phaf

I don't have much experience in PCR. I will try my best to help you out.
you can get short fragments and your pcr is quite long.
_ try decreasing the Tm.
_ extend the extension time

first of all, make sure your primers are right. Even that one base doesn't match will give you no product.

#3 kant0008

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Posted 06 December 2004 - 09:24 PM

Hi!:rolleyes:

You say you get a product of about 80bp- is it a primer dimer? Are your primers complimentary/ self-complimentary? If they are, they could be binding to each other, and so not leaving enough primer available for the reaction. In that case re-design primers.
As far as polymerases go, Hotstar from Qiagen normally works well for me, and people also suggets Omniscript for RT (I myslef haven't tried it).

Also, if you could let us know what you've tried in terms of varying the reaction conditions we could perhaps give more suggestions.

Good luck!

#4 phaffia

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Posted 09 December 2004 - 06:35 PM

Thanks very much netnus and kant0008 for your suggestions!

Netnus: I have tried toggling with the annealing temperature and extension. Didn't work.

And yes kant0008, I believe the PCR products that I've seen were primer dimers as I ran another round of PCR without the template. Those bands (of the same sizes) appeared. So...hah....I'm back to zero.

Have designed other primers that can give me various fragments of 100-200 bp and found one pair that works well at generating a 150 bp fragment. Used that forward primer and another reverse primer (that I've used for RT-PCR) to see if I can generate another 2.4kb fragment within the same region. (Reason I chose these primers is because previous results indicate that these primers worked.) This time, a fragment size, slightly smaller than expected, was observed; I got a 2kb band. Can a region within my template be difficult to amplify [maybe the possiblility of having a tight secondary structure (hairpin loop) present]?

Thanks again!

phaf




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