are you getting another fragment of 0.6 kb along with 1.5 kb fragment??? if so then surely your gene contain the restriction sites for one of the enzyme that you have added in forward or reverse primers, have you cheeked the restriction sites for these enzymes in your gene of interest.??? if not then check it using the NEB CUTTER.

I also had problems attaching GFP to one of my genes I finally solved the problem using overlapping pcr. I don't know if this has been suggested before as can't see original thread. It was the only method that worked for me and believe me I tried a few methods. Using this method you only have one fragment to ligate.

Thanks all for your reply.

I cannot do overlap pcr because my primers doesn't have overlap sequence of either genes. It only has restriction site-bamH1 with linker.

I have gene of 1.5kb and GFP -0.75bp.

The problem is I am not able to produce clone either with double ligation of gene and GFP or with triple ligation including double digested vector.

I do restriction digestion of gene from plasmid and amplify with phusion polymerase and proceed further.

my primers have high melting temperature above 80degree.

And the yield of gene after phusion pcr, is very low, this is another issue.

please, suggest me.

And I get so many colonies in self-ligation plate, which is not expected.

You can design primers to have overlap as in Soeiing method between 3' end of target gene and 5' of GFP

i.e. for your gene you use a 3' primer that has a bit of the 5' sequence of GFP at its 3' end and for your GFP 5' primer add some of your 3' sequence of your gene to the 5' end. This gives two pcr fragments that you can then put into one rxn using 5' gene primer and 3' GFP primer and this will produce some product which will contain both gene and GFP in correct orientation. This can then be ligated into vector. See attachment for better explanation. You will get a mixture of products in your colonies but should have some full length so colony pcr should identify these. Hope that is clearer.

http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction

I will suggest to perform overlapping PCR with NEB Q5 high fidelity DNA Polymerase, which have 100x fidelity than normal Taq polymerase. GFP gene is easily fused into the gene of interest through overlapping PCR. You can re-design your primers with at least 20 nucleotides overlapping with each of the genes. The final product is easily clone into the vector. Do remember to include your RE site into the 5' and 3' end. I personally felt that overlapping PCR is much more simple than RE digestion. Good Luck.

Thanks all for your suggestion.

Even after using high fidelity phusion polymerase to amplify my gene of 1.5kb, I feel its not getting amplified with temperature gradient ranging from 55-70degree.

primers are designed according to the gene sequence. please, find the attached pics of gene after phusion pcr.

GFP phusion pcr pic.

please suggest your opinions.

Hi Megha Achar,

Are you using phusion to do overlapping PCR? I used iProof HF polymerase (bio-rad) before, but failed to get correct band size. The problem was solved when i using Q5 HF polymerase (NEB). The primer annealing temperature can be easily determined by using NEB Tm Calculator (https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator).

The ladder do not have the size, can't really analyse the gel.