Hi Madhuram,

In case that your strategy involves sub-cloning into another vector due to the fact that you have chosen the two fragments and primers B and C to introduce a mutation, I suggest that you familiarize yourself with the Gibson assembly technique. Link below:

https://www.neb.com/products/e2611-gibson-assembly-master-mix#tabselect2

I have used this many times and it is such a time saving tool:

Just open your targeting vector, do your separate PCRs, elute those from the gel and put everything together and add the mix. Only order the assembly mix, you can use your own competent bacteria etc. You will find that you can do so much in one go without having a second round of PCR. Read in to it I highly recommend it. I think it is the only leap forward in cloning together with synthetic DNA fragments for many years.

Best,

Stephan

For me, following protocol worked quite well:

~50 ng PCR I (Primer AB)

~50 ng PCR II (Primer CD)

in 50 µl PCR-reaction

10 cycles with an annealing temperature that corresponds to the BC overlap

(however I worked with q5 polymerase and used an annealing temperature of BC-overlap +3°C)

after 10 cycles add Primer A+D (5 µM each)

continue PCR for 25 cycles (adjust annealing temperature to AD primer-set)

I got lots of the desired PCR product, which I purified by electrophoresis and gel extraction.

Don't hesitate to ask if you need any further info!

Thanks Andreas: I want to know one more thing size of the two initial products are 314 and 854 bp. So if i take 50 ng of each there will be difference in copy number. As product smaller size will be in high no than the longer one. Will this effect overlap pcr. or i should take equal copy no of these product.

Second thing how u decided annealing temperature for initial 10 cycles in overlap pcr 

You'll maximise yield when you work with equimolar amounts of PCR I & II (in your case that'll be about 20 and 50 ng, respectively). However, I don't think that this is the most critical step.

I calculated the annealing temperature based on the BC overlap (in your case the 21 bp complementary region). You'll hardly get any PCR product, if your annealing temperature is too high. Of course you can work with (commonly used) 60°C,

but then you'll need more than 25 cycles in the subsequent PCR step with primers AD (because of the low yield in the first 10 cycles).

Hi Andreas:

I got results in Overlap  PCR. But there were lot of non specific bands present along with my desired product. So i gel eluted desired product and after that i did put next PCR to amplify the product by using primers of extreme ends by using gel eluted product but there was no amplification....for the pcr i used approximately 40 nanogram of previous overlap PCR product. ..What changes i should do in PCR to get the amplification of desired product...

 Hi, Madhuram, 

Have you got positive result of above-mentioned trouble in getting amplification when you used OL PCR product ? If yes, please share the changes that you have made, 

Hi Madhuram

Have you got the result of query mentioned on 25 sept 2015 regarding overlapping pcr.

An attentive user has made me aware of an error in the protocol. I forgot to specify the volume of the primer dilution. Here's the correct protocol with the final primer concentration:

For me, following protocol worked quite well:

~50 ng PCR I (Primer AB)

~50 ng PCR II (Primer CD)

in 50 µl PCR-reaction

10 cycles with an annealing temperature that corresponds to the BC overlap

(however I worked with q5 polymerase and used an annealing temperature of BC-overlap +3°C)

after 10 cycles add Primer A+D (0.5 µM final concentration each)

continue PCR for 25 cycles (adjust annealing temperature to AD primer-set)