Hi Nazmiara,

There's quite a lot of questions you have here, and to provide a full answer will be difficult. I'll try to answer what I hope is the main question you have here, and see where it leads us.

For the primer design for the overlaping PCR. I will just focus on the joining of your GOI to the eGFP for now. The principal holds for which ever parts you are joining. :

for GOI: you want to design a partial primer that bind to the 3' end of your gene stopping at the codon before the stop. Make sure it has a Tm of 55-60oC, this will be the start for a reverse primer. So you have:

5'ATG--------------------------------------TGA   GOI forward strand

                                                   3'xxxxx          Partial Reverse Primer

for eGFP: you want to design a partial primer that bind to the 5' end of your gene starting at the ATG start codon. Again make sure it has a Tm of 55-60oC, this will be the end for a forward primer. So you have:

5'ATGaaaaaa                                                       Partial Forward Primer

3'TAC--------------------------------------ACT    eGFP reverse strand

So far as normal.

Now you design the reverse compliments to both partial primers, so:

5'yyyyy             Reverse Compliment of  Partial Reverse Primer

3'xxxxx             Partial Reverse Primer

and 

5'ATGaaaaaa    Partial Forward Primer

3'TACbbbbb     Reverse Compliment of Partial Forward Primer

Now you connect the two primer sets together so:

5'yyyyyATGaaaaaa   Forward Primer for eGFP gene

3'xxxxxTACbbbbb    Reverse Primer for GOI

Running these primers in PCR reactions with  forward or reverse primers as appropriate for the gene will result in fragments that have complimentary overlapping ends. These fragments can then be combined and a second round PCR run with the outer PCR primers. So:

                     PCR 1                                                          PCR 2

5'--------->                                        5'yyyyyATGaaaaaa>

5'ATG------GOI------yyyyyTGA                 5'ATGaaaaa-----eGFP----TGA

Hi Nazmiara,

There's quite a lot of questions you have here, and to provide a full answer will be difficult. I'll try to answer what I hope is the main question you have here, and see where it leads us.

For the primer design for the overlaping PCR. I will just focus on the joining of your GOI to the eGFP for now. The principal holds for which ever parts you are joining. :

for GOI: you want to design a partial primer that bind to the 3' end of your gene stopping at the codon before the stop. Make sure it has a Tm of 55-60oC, this will be the start for a reverse primer. So you have:

5'ATG--------------------------------------TGA   GOI forward strand

                                                   3'xxxxx          Partial Reverse Primer

for eGFP: you want to design a partial primer that bind to the 5' end of your gene starting at the ATG start codon. Again make sure it has a Tm of 55-60oC, this will be the end for a forward primer. So you have:

5'ATGaaaaaa                                                       Partial Forward Primer

3'TAC--------------------------------------ACT    eGFP reverse strand

So far as normal.

Now you design the reverse compliments to both partial primers, so:

5'yyyyy             Reverse Compliment of  Partial Reverse Primer

3'xxxxx             Partial Reverse Primer

and 

5'ATGaaaaaa    Partial Forward Primer

3'TACbbbbb     Reverse Compliment of Partial Forward Primer

Now you connect the two primer sets together so:

5'yyyyyATGaaaaaa   Forward Primer for eGFP gene

3'xxxxxTACbbbbb    Reverse Primer for GOI

Running these primers in PCR reactions with  forward or reverse primers as appropriate for the gene will result in fragments that have complimentary overlapping ends. These fragments can then be combined and a second round PCR run with the outer PCR primers. So:

                     PCR 1                                                          PCR 2

5'--------->                                        5'yyyyyATGaaaaaa>

5'ATG------GOI------yyyyyTGA                 5'ATGaaaaa-----eGFP----TGA

Dear Nazmiara,

It sound easy. You open your vector with two restriction enzymes that do not cut your gene of interest. It will be even simpler if one of the enzymes is a blunt end cutter. Do not de-phosphorylate your vector.

Amplify your gene of interest. Design the forward oligo with ca 3 nucleotides followed by the restriction enzyme site, a Kozak consensus, your ATG and a stretch of priming nucleotides. If your 3' site is also an enzyme site with overhangs start as above, 3 nucleotides followed by the enzyme site and instead of the ATG, the gene-specific sequence starts (in reverse) with the last codon preceding the stop codon. In case of a blunt end side just start with the (in most cases) 3 nucleotides to complete the restriction site. The advantage of one blunt end site is the shorter primer and the need to cut the PCR product with just one restriction enzyme. Before ordering your reverse oligo you have to check the open reading frames. Eventually you have to add 1 or 2 nucleotides between restriction site and gene specific nucleotides. Doing this, avoid to generate a stop codon or a very rare codon.

Cut your GOI, purify the DNA and do a ligation.

Since restriction enzymes do not cut efficiently at the end of a fragment, the recommended number of nucleotides added differs from enzyme to enzyme. The BioLabs website gives some data

Thank you so much!!! I have designed primers and let's see hopefully it will work.