Dear Renzo,

Please find attached, the strategy we used to generate a construct using 3 genes of interest.

To put the strategy simply:

First, design primers: the gene located between the other 2, should contain homology arms for both adjacent genes. The first and third genes do not need to contain any homology overhang with the second gene.

Second, you will have to perform 3 rounds of PCR and product purification:

In the first round of PCR, amplify each gene of interest in separate tubes, lets call them tubes A-C as the first, second and third gene of interest.

Purified PCR products A and B will then be the templates for the second round of PCR (D). That reaction will use the forward primer for the first gene of interest and the reverse primer for the second gene of interest. Run that sample on the gel to visualize successful stitching and purify.

Then the third round will use the purified PCR product from reaction C and D to add the third gene of interest to the constructed first and second gene of interest construct. Primers for this will be the forward primer for the first gene and the reverse primer of the third gene. Again visualize on a gel and purify.

Lastly, remember that the homology arms on the primers for the 2nd gene, will be at the 5' end of the primers, and make sure the sequences are in the correct orientation.

Hope this helps

Dear Renzo,

Please find attached, the strategy we used to generate a construct using 3 genes of interest.

To put the strategy simply:

First, design primers: the gene located between the other 2, should contain homology arms for both adjacent genes. The first and third genes do not need to contain any homology overhang with the second gene.

Second, you will have to perform 3 rounds of PCR and product purification:

In the first round of PCR, amplify each gene of interest in separate tubes, lets call them tubes A-C as the first, second and third gene of interest.

Purified PCR products A and B will then be the templates for the second round of PCR (D). That reaction will use the forward primer for the first gene of interest and the reverse primer for the second gene of interest. Run that sample on the gel to visualize successful stitching and purify.

Then the third round will use the purified PCR product from reaction C and D to add the third gene of interest to the constructed first and second gene of interest construct. Primers for this will be the forward primer for the first gene and the reverse primer of the third gene. Again visualize on a gel and purify.

Lastly, remember that the homology arms on the primers for the 2nd gene, will be at the 5' end of the primers, and make sure the sequences are in the correct orientation.

Hope this helps

Hello,

I assume you are trying to join 3 pcr product of might be part of some full length gene. So you can go through mathew protocol as it is very well described for primer design.

Any further help find attachment for protocol of pcr.

Dear Cortez,

You can find a good information regarding primer design and amplification here:

Article Evaluation of pEASY- Uni Seamless Cloning and Assembly Kit t...

Good luck,

Dineshkumar Krishnamoorthy

Sorry it was a protocol I wrote for myself and therefore I knew what Caax was used for, I didn't think I was going to share it with others.

Anyway, the Caax domain is a motif that sends the GFP to cellular membranes. GFP without the CAAX domain will broadly express throughout the cytoplasm. So the alternative primers with and without the Caax domain was alternate ways to detect the GFP signal as we were unsure how sensitive the sensors we were creating would or could be. We had to use different templates for the GFP amplification with and without the Caax domain as most GFP proteins do not contain the Caax domain.

Therefore you need to determine whether you want your GFP protein to localise on a membrane or be broadly expressed throughout the cytoplasm.

If you need any other help or advice, I'm happy to help.

Good luck

Then you won’t need the CAAX motif in your sequence!

Matthew de Cruz ,

We have been opted your strategy for primer designing to merge three fragments of interest. Since, our second fragment (the centre fragment) Tm is higher than 80 degree, therefore, how to set up our PCR programme. I mean how could specify annealing temperature of the primer.

Suraby Ej firstly, is the TM 80C for the entire primer or just the homologous part that will bind your template DNA?

You should only take into account the annealing temperature of the homologous region to your template. If this is true, then you should do a Ramp PCR, which is basically 10 cycles at the temperature for your homologous region and then 25 following cycles at a higher temperature. This will allow the first 10 cycles to prime to your DNA template and amplify your Gene of interest and then once you increase the temperature your PCR product which includes your homology arm overhangs, will become the new DNA template for amplification.

Currently I have been amplifying cDNA for in vitro transcription, of which my primers contain T7 promoter overhangs. In my experiments, my overall primer TM is 71C, the homologous part of my primer has a TM of 59C. Therefore for the first 10 cycles our annealing TM has been 55C and then the following 25 cycles are 62C. A general rule is to reduce your annealing temperature roughly 5 degrees to allow a little non specific binding.

Also I would avoid your TM being around the same temperature for your DNA polymerase, assumably that would be 72C.

I hope this helps,

Matt

Matthew de Cruz Thank you so much for your prompt clarification.

The Tm for entire primer is 80C. Tm for forward and reverse primers of gene of interest is 75C and 66C respectively. I used 68C for amplification of GOI in first round PCR.

However, Tm for homology part is 62C. Shall I use annealing temperature of 62C for first 10 cycles of ramping PCR and then 25 following cycles at a temperature of 68C?

Thanks

Suraby

The forward and reverse primers TM are drastically different. You want to aim to have a TM for both forward and reverse within 1-2 degrees of each other, so both can efficiently bind the template. If your annealing temp is 68C I don't believe the 66C reverse primer would bind well.

When trying to SOE 2 PCR fragments together, the temperature of the homology arms fusing those 2 fragments together, aren't that important, as you'll be using the forward primer for PCR product 1 and the reverse primer of PCR product 2, so you have to tailor the annealing temperatures for those For and Rev primers.