Hi everyone,
I am currently trying to splice two DNA fragments together by OE-PCR. I have one fragment which is around 1400 bp and the other is around 100bp. The two fragments have 20 bp in common.
In my PCR reaction, I add 10ng/ul of both 1400bp and 100bp. I then try to splice and amplify them into 1500bp fragments by running 34 cycles. But after I ran my PCR products on the gel, I only got a wide clear band ranging from 250 bp to 1000bp.
Here are some details :
- I thought this was because the primers were bound to the wrong place and amplified the wrong segment. But after I changed the primers it was still the same.
- There was actually a very faint band around 1500bp under UV every time I did the OE-PCR. I tried to extract the DNA but the yield is very low.
Thanks for your kind help.
I think that this may be a problem with over amplification not with the OE process. You are running 34 cycles of pcr which could lead to 8x10exp9 copies of the product. This is too much and the product may be self annealing and forming smears of amplified product. Try setting up the pcr in tubes and remove a tube from the pcr machine every 5 cycles.you may find a stronger amplification at 10 or 15 cycles
Thanks, Paul. I will try to cut the cycles down in my next experiment and see if the amplification is improved.
I assume that after the initial annealing of the 2 fragments you extend the product to form a double stranded template so that the template does not fall apart on the first round of pcr as the 20 base overlap may have a lower annealing temperature than the primers
As your fragment sizes are quite different, for the same amount of DNA, there will be more fragments of the smaller band size present. So, you may consider that while you are adding the template in PCR mix. Hope that helps.
Dear Zhang,
1. As both the DNA fragments are of different sizes, do consider adding equimolar concentration (In your case, for 1ul of 1400bp fragment (100ng/ul), add 7ng of 100bp fragment).
2. the overlap sequence size should always be more than the primer size (I recommend more than 50bp). Here in your case, the 20bp overlap is too small as Paul Rutland said; the two fragments may fall apart during the initial rounds of PCR.
Thank you Shomoita and Vinay. After I changed the overlap sequence from 20 bp to 26bp the OE PCR worked. Now I have a band around 1500bp revealing clearly in front of a smear. I will do further optimization to reduce the smear.
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