reampllification of pcr product - reamplification (Dec/02/2006 )

hi,
i have done 20ul pcr reaction for 1.3 kb band , i loaded 18ul of pcr sample out of 20ul on agarose gel i got that 1.3 kb band ,2ul which was remained frm that 20ul reaction i have taken 1ul frm that 2ul pcr sample and 199ul wated added in that so it was diluted then again i have done pcr for same condition for reamplification of that product but i got only high concentrated smear on agarose gel so what ll be the resone why i m not getting band pls tell me

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From your description, it sounds that you did not clean up your first PCR product before you used it for the second PCR amplification. Due to the excess primers and dNTPs, you got smear on agarose gel.
The solution is, load everything on your gel and do gel purification of that product before you use it for the next round of PCR reaction. Alternative, if you are very sure that your first PCR give you the right product, then you can do PCR clean up before the next round of amplification.

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thanks a lot for ur valuable suggestion

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how can i clean up pcr product before d next round of amplification

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i use the PCR clean-up from qiagen. It's part of their gel extraction kit.

V

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Another reason for getting a smear is using the same primers in the second PCR. I would suggest performing a nested PCR, using outer (flanking) primers in the first reaction, then diluting and carrying out the second reaction with the actual primers you want to use. This should definitely help the smear.

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How does using the same primers create smear? I have had few problems with reamplifying a PCR product using the same primers.

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I think it is due to the excess primers. I am not very sure but it didn't happen to me previously.
To girish.jln, even after the PCR clean up step, I would suggest that you do different dilution to your cleaned PCR product, eg. 1:10, 1:100, 1:1000, etc.

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When you use a pcr product to make another pcr without cleaning first you will have not only more primers, but also more enzyme, MgCl2, etc and that contribute to the smear...you could:
1. clean it first
2. The second reaction add less MgCl2 and less primer and 1micro of the reaction.
3. use another set of primer for the 2nd reaction
have fun!!!hope that this could help

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hi girish,

u can run the PCR amplification rxn on the gel and when u visualize it on the UV u need to prick the center (from all sides) of ur band of interest with tip of the 'TIP' and dip this tip into another PCR rxn mix (20 ul volume) without template (this will serve as the template for ur second round of PCR). this serves well and also eliminates the elution extraction and purification.......! u may need to keep some replications u ensure the uniformity....! this has pretty well worked so u can try it out........

gud luk

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