Hi,
I want to get a pair of primers that contain a restriction site that would allow me to clone the PCR product afterwards. Which software can I use for this? Thanks in advance!
you do not need software just take the primers that amplify your product and add the restriction site at each 5@ end of the oligo. Then add 3 more bases ( any bases) to ensure good cutting with the enzyme. When you calculate the annealing temperature of the primers do not include the restriction site...only the target specific part of each primer
I personally use SeqBuilder(from DNAstar) which has an option for this. SnapGene also does this, I'm sure. ApE ("A plasmid Editor") is a freeware program that I *think* can do this, I don't like ApE personally but if you can't buy other software it might work.
you do not need software just take the primers that amplify your product and add the restriction site at each 5@ end of the oligo. Then add 3 more bases ( any bases) to ensure good cutting with the enzyme. When you calculate the annealing temperature of the primers do not include the restriction site...only the target specific part of each primer
Check the "Restriction close to the ends of DNA fragments" chapter in the appendix of any New England Biolabs catalogue to find out the number of bases you need to add between the primer 5'-end and your restriction site for the enzyme to cut efficiently. Otherwise cloning will be nightmarish.
Since you usually need to verify your PCR sequence before cloning, another approach is cloning your PCR into any T-vector, sequencing to verify it, then cut the plasmid. You can do without adding bases at the 5'ends then.
No, it would not work, unless you have written primer 2 in the direction 3' to 5'. If indeed you have written primer 3 from the 3' end (cgtcc...) to the 5' end (ggatccnnn), then it would work. But beware when you order the primers to put them in the right order.
At any rate, restriction enzyme sites should in general be added at the 5' ends of primers, whether the "upper" or "lower" primer.
Well, the BamHI site is a palindrome (reads the same in the 5' and 3' directions), so no fuss there. It is correct. But yes, in those rare cases when RE sites are nonpalyndromic, they have to be added in the same direction than the primer.
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