Hi Deepthi,

Hairpins form when your primer is able to form a number of base pairs between two separate regions along its length after it folds back on itself. An example of such a primer is:

5' AC(GTGCCACG)ATTCAA(CGTGGCTC)AG 3'

The intervening region (ATTCAA) is amenable to bending to allow the two bracketed regions (in parentheses) to come into close enough proximity to base-pair, forming hydrogen bonds that stabilize this secondary structural element.

To avoid hairpins when designing your primers, you can use software products that companies such as IDT DNA and Sigma build into their ordering pages. Alternatively, freely available programs such as the Northwestern University web-based software, OligoCalc, will also be useful to you.

http://www.basic.northwestern.edu/biotools/OligoCalc.html

They will also help you avoid primer dimer formation, another common problem.

Good luck!

Hi Deepthi,

Hairpins form when your primer is able to form a number of base pairs between two separate regions along its length after it folds back on itself. An example of such a primer is:

5' AC(GTGCCACG)ATTCAA(CGTGGCTC)AG 3'

The intervening region (ATTCAA) is amenable to bending to allow the two bracketed regions (in parentheses) to come into close enough proximity to base-pair, forming hydrogen bonds that stabilize this secondary structural element.

To avoid hairpins when designing your primers, you can use software products that companies such as IDT DNA and Sigma build into their ordering pages. Alternatively, freely available programs such as the Northwestern University web-based software, OligoCalc, will also be useful to you.

http://www.basic.northwestern.edu/biotools/OligoCalc.html

They will also help you avoid primer dimer formation, another common problem.

Good luck!

You can use primer 3 online tool for designing PCR primers.

I'm sorry, the underlines seem to have shifted after I posted my answer! I have now indicated the hairpin-forming sequences with brackets/parentheses.

I use this site. It always works for me.

http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi

Hello Deepthi,

You just need to remove or add nucleotides at the 5' or 3' end (ranging from 18 to 21 nts) to avoid secondary structures. We generally prefer sigma oligo evaluator for designing any primers or oligos and finally we would check with Primer express software before ordering!! That works very fine :) Good luck

You can always use a number of software to make primers for your target sites. You need to check at which temperature it actually forms, if it is forming at lower temperature from that of annealing temperature then you does not need to worry. Such type of problem does occur during cloning a specific region. These problems can also be sorted out by adding PCR enhancer solution in your mastermix. Good luck.