how to synthesize the pcr primers - (Jul/08/2008 )

hi all
i wnat to design the pcr primers for amplification of desired gene
i am not able to synthesise these primers with online softwares and fast pcr software also.., iam missing some something and i may be doing wrong
the results were giving from middle of sequence and so many parts of sequnce ., but i want to amplify the full gene sequence
could anybody please help me step by step procedure how to design primers with any one soft ware
please kindly help me out
i need help
thank you

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Hi vision, the best way to design primers for full lenght amplification is to do it the old way, ie. manually with paper and pen
Design your primers to include ~20-27 nt of both 5' and 3' end of your desired sequence. Then use primer 3 (or any other software) to confirm they work. Sometimes the software will tell you that your primers wont yield any product and you will just have to try them.

Good luck!!

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hi doctor
thank you
i will fallow this
can i know how to modify the primers if they were wrong
suppose the primers that i have synthesized were hair pin or high gc rich then how i have to procedd next to fallow and how to make it to work out
please tell briefly
i need help
thank you

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Hi

As Doctor suggested you can design primers at the 5' and 3 flanking sites.
Select sequences in such a way that ur start and stop codons will be included in the amplicon. Use around 22-26 bp stretches as the primer region.
I normally use the Oligo Calc: Oligonucleotide Properties Calculator at
http://www.basic.northwestern.edu/biotools/oligocalc.html for checking primer properties ( Tm, GC content, dimers, hair pain etc)

Make sure the Tm of these primers are compatible ( difference between the Tm should be kept below 3-4 degrees)

I generally try to maintain 40-60% GC content for the primers.

If ur gene has high GC content , you can try using DMSO or PCR enhancers like betaine.

all the best

Avinash
http://aviprem.gq.nu

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