how do we determine primers? basic question

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nananvatip
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how do we determine primers? basic question

Post by nananvatip » Sun Feb 26, 2006 3:21 am

I have no experience in Molecular biology and would like to get into the field. I am reading about PCR and I dont understand how we can know which primers to use and how do we make primers?
Thanks

shamim khaja
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Primer

Post by shamim khaja » Mon Mar 06, 2006 1:49 am

primers are short stretch of nucleotides whose sequence has been picked from the template strand.
We have a "forward primer" and a "reverse primer". The forward primer has a sequence similar to the template strand for 5'--3'. Where as the reverse primer has a sequence complementary to the strand 3'---5'.
To design a primer first u have to know the sequence of your template strand i.e. the sequence of ur desire gene.
hope that has helped u
Satisfaction of one's curiosity is one of the greatest sources of happiness in life.
Linus Pauling

nananvatip
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Post by nananvatip » Tue Mar 07, 2006 1:23 am

Thanks a lot, this helps.

kk
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Post by kk » Thu Mar 09, 2006 8:57 pm

I my lab we follow these criteria:

1. primer length should be 18-24 base
2. GC content should be 55-60%
3. the melting temperature of the two primers should be similar
4. G or C are preferred, especially at the 5' end of the primers

5'-XXXX.......-3'

- the first and second (from the 5') should be both G or C
- at least 3 G or C in the first 4 base

5. avoid runs of 4 bases the same (e.g. AAAA)
6. check primers for hairpin or dimer formation (shouldn't form)
7. blast the sequence

robertlee_ioz
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Post by robertlee_ioz » Mon Mar 13, 2006 2:37 pm

thanks for the question and answers especially that form kk.
I am a beginner and this helps me :D

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