Hi there,

Are your fragments already overlapping?

If no you need to create the overlap between the 2 with a first PCR with dedicated primer sets and then perform the proper overlapping pcr using the two fragments as Template and only the outer primer set for amplification of the expected product.

If yes, only the second pcr is needed. The protocol is similar to the classical pcr. As first you need to encourage hybridization of the two fragments, you may run the few first cycles without outer primer set and then add them to allow exponential amplification of the fusion product.

Personnally starting from overlapping fragment, I use 50ng of the mix of the 2 as DNA Template. Run 2 cycles without outer primers and then add them for 33 more cycles (denaturation 30 sec at 98°C, hybridization 30sec 55°C, elongation 15sec per kb).

Hi Dominique,

thank you for your explanation. Actually my fragments both should be overlapping. I did the first PCR reaction with:

Denaturation 98    1 M

Subsequent    93     20 Sec.

Annealing        56     30 sec.

Extension         72 2:30 sec   x 10 cycles

but it does not work. do you think the gene size that I want to overlap will effect on the reaction result?

Hi again,

10 cycles is not enough for visible amplification if you start from ng quantities... So you can't say it's not working at the moment... Furthermore, your elongation time is far too much for Phusion amplification on purified Template (15 sec per kb is OK, up to 30 sec for difficult Template).

thank you again,

I thought the same as you for 10 cycles and it might not enough to show a remarkable result. however I did the next step of the experiment in the presence of the primers on the following conditions:

98    1M

96    20 SEC.

60    20 SEC.

72    2 M

F E 72 3 M

* 35 Cycles

and I got negative result. ( I got two band for the same sample!!)..

but do you thing the real defect was on the elongation time?

No.

How much of each DNA do you introduce? Do you respect 1:1 molar ratio?

How large is the overlap and what is the Tm of this isolated sequence? It might be a problem of hybridization...

What are the sizes of the bands you get?

I did different DNA ratio, 1:1 , 4:1,  4:4 ratio.

20 bp as an overlap .

I have got the same band size before the overlapping.

20bp overlap  migh be a bit short. I usually go for 30 to 40bp.

Hello, I am new in Overlapp PCRs but this two month I been working a lot and I learn a lot about it. In my case the overlapp sequence is 24bp and works very well. Something that had changed my life was reducing the amount of primers used in the overlap PCR. Found attached the figure.

As it was said before, my protocol is very similar.

Gel purify each fragment. 

Mix in 100 ng each (equal molar ratio) in final volume of 50 uL using phusion enzyme (0.5 uL/2.5U). MAke a PCR without primers for 20 cycles using annealing at 56C and extention for 40 seg.

Then take 10 uL of this "fill in" reaction to a new 50 uL PCR with primers for 30 cycles in the same conditions. BE CAREFULL of the amount of the primers. I use 1 uL of 2.5 uM of each primer, or even less. See the picture attached. Wish you all the best!!!

Hi Martin, 

thank you so much for your explanation and I will take in you point as well.. Thank you and wish to you all the successful experiment :)

Hello Martin,

I was just following suggestions for OE- PCR. I just wanted to know when you said 1ul of 2.5uM reaction you meant: taking 1ul from 2.5uM primer stock and adding to 50ul reaction?  

Hi Bhakti,

I think he meant taking 10 ul of the step A PCR reaction and make step B ,50 ul total, (1 or less of 2.5um each primer, 10ul A, h2o,  phusion polymerase and dNTPs).