Getting an 8 kb amplicon by PCR may not be trivial; have you checked with a different control (a reaction that would yield an 8 kb or longer amplicon) that your conditions are OK before proceeding to SOE?

Also, why don't you try Gibson assembly instead?

Dear Vedita,

Have you checked if your primers somehow match another part of the sequence that you want to amplify? Maybe the primers attach at a point further down the sequence so you get a smaller fragment eventually. In that case you will need to either change your primers or increase the temperature so as to increase specificity. 

Also if you would be willing to re-consider your technique (and if it's relevant to what you try to clone) I would suggest following this method suggest by Zhang 2012 for an easy way of overlapping different segments of DNA together. 

DOI: 10.1093/nar/gkr1288

Hope that helps

Nina

Hello Alejandro, 

I have not tried Gibson assembly kit. I thought overlap strategy might work well. 

The overlap region is of 30 base pair. Should I go for a single PCR? What changes should I make? What controls should I take? 

I am new to this field, and recently started working with overlapping primers.

Please help. 

Dear Nina, 

I have checked the specificity of the primers, they do not attach to other parts of the gene.

I have amplified two, 4 kb segments separately and have also gel purified them using QIAGEN kit. Now I need to join them and make a 8 kb amplicon. And this part is troublesome. 

I am attaching the gel image. Please have a look. The ladder is of 10kb. I am getting multiple nonspecific bands but not 8kb desired band.  

Given that the problem seems to be complicated, I would suggest you have a read on the article I sent you for a faster and more effective method of DNA recombination. The method uses a bacterial extract from a specific strain of E.coli to be used as the 'enzyme cocktail' for efficient ligation. That's what we use in my lab, where we clone various combinations of genes and it seems to work every time. We no longer use the PCR method for overlapping because it would bring up all sorts of issues. As long as you have the pieces you want to join, this bacterial extract and a t4 ligase buffer, you can perform the ligation in 20 minutes. 

Thank you, Nina, for your suggestions.  Will this work for more that 4 kb fragment size? 

By the way, I will read the suggested paper also. 

Nina, that procedure looks very attractive. Two questions, 1- Are you aware of any alternative to the protocol for preparing SLiCE extracts where lysis does not depend on a proprietary reagent? and 2- Where can I order the PPY strain?

For Vedita: Yes you can use it with any fragment big or small, from my experience I have performed this technique for 7-8kb fragment and it has worked. 

For Alejandro: 1- You can perform the lysis using the standard techniques i.e freeze-thaw, lysozine and sonication or french press, we didnt use proprietary reagents 2- you can contact the authors, they will be happy to provide you with the strain

Hey Nina and Alejandro, 

It would be helpful if you guys can suggest me points I should keep in mind before starting for Gibson assembly of 3 fragments, 5 kb each. I don't want to insert them into a vector but want then spliced together.

Dear Kyle, 

The PCR reaction condition that I used for above gel picture of overlap PCR is

 I am performing 15 cycles of PCR without primers at 60oC annealing for 20 minutes. and Extension at 72oC for 8 minutes. 

Then I add primers in the PCR tubes and again set for 32 cycles,60oC annealing for 1 min, and Extension at 72oC for 8 minutes. 

The denaturation temperature is set at 950C for 1 minutes and final extension is set at

72oC for 15 minutes.  

I use HerculaseII from Agilent for all overlap PCR. I use 50 pM primer concentration in a 50uL reaction.