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design primer


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#1 saad

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Posted 05 March 2009 - 03:44 PM

Hi
I haven't a good experience in primer design and I would like to design a forward and reverse primer for insert in plasmid to do PCR
I have separately 3 inserts(domains) from DNA in a plasmid so I hope to help to design a FWD and REV primers for each.
is it necessary to include the restriction sequence in the primer.
Thank you so much in advance

#2 perneseblue

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Posted 05 March 2009 - 05:14 PM

Hi
I haven't a good experience in primer design and I would like to design a forward and reverse primer for insert in plasmid to do PCR
I have separately 3 inserts(domains) from DNA in a plasmid so I hope to help to design a FWD and REV primers for each.
is it necessary to include the restriction sequence in the primer.
Thank you so much in advance



could you explain what do you want to do with the three PCR fragments?

Are they to be ligated into a single plasmid? Are they to build a complex fusion gene?

In general, adding restriction sites to your primers, allows you to perform stick end ligation. And sticky end ligation is easier than blunt end ligation.

Of course there is TA cloning which does not use restriction site, but it requires you to have a TA cloning vector.
May your PCR products be long, your protocols short and your boss on holiday




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