I have done this several times, joining together either two or three PCR products. You need a 40bp overhang on one of the PCR products that is complementary to the end of the adjacent PCR product (add this with your initial primers). If you are trying to PCR all three fragments together in one reaction, try instead to attach two and then add the third one on in a separate reaction - this always works better. Additionally, you need to allow the PCR products to anneal (at higher than normal annealing temp so you don't get nonspecific hybridization along the long PCR products that are non complementary) (I use 68 degrees), and fill in to make a double stranded product BEFORE adding the terminal primers. So basically have everything in your PCR reaction EXCEPT primers, do 2-3 cycles of annealing and extension, and then you should have double stranded full-length products. Then add your primers to the distal ends of the fragment and do a normal PCR reaction. I have not had luck with any enzyme besides Taq for this. Hope this helps!

Hi Praveen

The approach of joining the fragments may be possible if your fragments have complementary regions at the end, which if compatible ..may be possible to ligate using primer extensions instead of ligation ....how ever you may have to take care of the 3'-OH for priming .....

bye

Hi, Thanks a lot. But I tried using this technique but during pcr using three fragments as template , I always get many bands and never get desired band..I have no idea whats wrong with it..If you can give full protocol and small hidden tricks , I would appreciate very much..Thanks..

Hi Praveen

the best thing will be to work with equimolar amounts of the fragments and even if you are getting multiple bands...on the gel...try to gel elute/ band-stab PCR of the desired fragment ....it should work.......But can you elaborate why you are trying this approach and not ligation of the fragments...

bye

Hi Praveen

I do not know the details of your workflow. However I'll tell what I did. I amplified a gene from plasmid 1 and another gene from plasmid 2. During these amplifications be sure to use high fidelity polymerase...not regular Taq using designed fusion primers. Also make sure the the overhangs of both the segments are long enough I had 30-40nts so that they can hybridize.

Finallly amplify both the segments with the distal end primers. you'll need to use regular Taq if you need to do Topo cloning.

Hope this helps

Good Luck

Hello,

Is Gibson Assembly the procedure you are trying to follow? : http://neb-online.de/Gibson_Assembly.pdf

I have done this several times, joining together either two or three PCR products. You need a 40bp overhang on one of the PCR products that is complementary to the end of the adjacent PCR product (add this with your initial primers). If you are trying to PCR all three fragments together in one reaction, try instead to attach two and then add the third one on in a separate reaction - this always works better. Additionally, you need to allow the PCR products to anneal (at higher than normal annealing temp so you don't get nonspecific hybridization along the long PCR products that are non complementary) (I use 68 degrees), and fill in to make a double stranded product BEFORE adding the terminal primers. So basically have everything in your PCR reaction EXCEPT primers, do 2-3 cycles of annealing and extension, and then you should have double stranded full-length products. Then add your primers to the distal ends of the fragment and do a normal PCR reaction. I have not had luck with any enzyme besides Taq for this. Hope this helps!

I am doing a very similar reaction like April but I am not adding any distal primers at all. In my hands, at least, adding those primers lowered the yield of the final product. I would instead simply mix equimolar amounts of the two large PCR fragments that hybridize at their 3'-ends (25-40 bp overlapping region. I check with the free online software Netprimer that the Tm is above 65 degrees) and do a "primer extension reaction" rather than a "real PCR". Good luck!

For projects like this I strongly recommend Gibson assembly (already mentioned by Gustavo). You can join several fragments with overlaps of 20 - 40 bases in one single reaction and still get a decent number of clones on the plates.

http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html

Dear April,

Could you share a complete protocol you followed, mentioning exactly what were the PCR parameters and Tm of your fragments. I am unable to get any amplification in my overlapping PCR

Whether 20nt overlap fragment is sufficient for fusion pcr

Does anyone have a good protocol to go by?

i have joined two fragments in PCR. First I put the reaction without primers and after the reaction is over I add primers and again perform the reaction. I keep the condition same for both the reactions, except annealing temperature. In my case overlap sequence generally lies between 18 nt to 26 nt. Fragments sizes were 1 kb and 0.5 kb approx. but with higher fragment sizes or fragments with large size difference some problem might occur for annealing.

Try Gibson assembly. It should work fine for up to 3 fragments but it becomes less efficient as you increase the number of fragments.

Dear April Pawluk,

Can you please share me the detail protocol? I want to add 3 prc product with 20-35 bp overhangs and all PCR product is in the range of 350 to 1500 bp size.

Thank you.