I have been setting up overlap extension PCR to join four fragments of size 500, 2140, 1300 and 500 base pairs.

I am adding equal amount of DNA in molar ratios and using Taq Polymerase with traces of Pfu.

I am obtaining the final 4200 base pair product (several times)

I have gel run the product and cut the band and gel eluted the same.

Next I have checked the concentration and run the gel of purified product and verified the correct size of the band.

However when I am using the product as a template in amount of 30ng to 60ng to scale up the product with the same terminal primers (used to obtain overlap extension product) I am not getting any amplification of correct size. The melting point of both the primers are checked via OligoCalc and within 2C. I have tried with GoTaq Green and Pfu Turbo but without result.

Also when I am tried to amplify the 2140 base pair fragment using a different set of primers I am seeing only a weak amplification- again all primers are checked for melting points and their is specific binding is verified using Snap Gene.

The same problem is reported by other colleagues from my and other labs also.

Really it is frustrating. Any clues?