Dear Researcher,
I am characterizing a domain of an arabidopsis gene and in order to do that, i am making internal gene deletion of around 300bp region, located inside of the gene of interest. Since i am using conventional method in my laboratory, may i know is it acceptable if i replace the domain of interest (~300bp) with a single restriction enzyme site (for eg BamH1) and analyse these deletion construct for its functions? In detail, I will design two sets of primers spanning front region and back region excluding the domain of interest, At the 3'end of my front primer and 5' end of back primer i introduced BamH1 restriction site. After the amplification, both front and back fragment is ligated and the resulting construct will be sequenced to make sure no amino acid shift.
I will appreciate if there is any reference/publication attached for these method. Thank you!!
Replacing the domain with a restriction site is acceptable, but perhaps a more straight-forward way is to use overlap or fusion PCR to join the two fragments. The first round amplification step is the same as the ligation method you propose, but instead of adding a restriction site in your primers, you tail your primers with 25-30 (in total) perfectly complementary nucleotides that match the flanking sequence of the region you want to delete. After gel purification, you simply add the two fragments together and use the outermost primers (no inner primers) to perform a "full-length" PCR to join the two fragments. The overlap region ideally will have an annealing temperature higher than the outer primers, therefore these fragments will anneal first and allow amplification of the fused product. You can check/purify your fusion product by gel electrophoresis before proceeding. This technique is amazingly easy, quick, and effective.
There are many overlap/fusion PCR tutorials on the web.
apologies, this is not my area of expertise and hence I can not help you.
Replacing the domain with a restriction site is acceptable, but perhaps a more straight-forward way is to use overlap or fusion PCR to join the two fragments. The first round amplification step is the same as the ligation method you propose, but instead of adding a restriction site in your primers, you tail your primers with 25-30 (in total) perfectly complementary nucleotides that match the flanking sequence of the region you want to delete. After gel purification, you simply add the two fragments together and use the outermost primers (no inner primers) to perform a "full-length" PCR to join the two fragments. The overlap region ideally will have an annealing temperature higher than the outer primers, therefore these fragments will anneal first and allow amplification of the fused product. You can check/purify your fusion product by gel electrophoresis before proceeding. This technique is amazingly easy, quick, and effective.
There are many overlap/fusion PCR tutorials on the web.
Thanks Alevtyna Yakushevska!!
Hi Timothy E Gookin, Thanks for you suggestion. Ya.i checked most references using that method for domain deletion analysis. Anyway, do you have reference of publication for using the method i mentioned? Because i already had made the contructs using that method so im looking for any references using that method to be included in material&method section. I could not find any of it. Thanks!!
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