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Designing PCR Primers for cloning

primer design cloning

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17 replies to this topic

#16 taiju

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Posted 17 April 2013 - 03:24 PM

So long as you include the coding sequence, there shouldn't be a problem with using primers that are outside the coding region. However, for the forward you don't want it to be too far from the promoter such that it falls off the sequence before reaching the ATG.


Awesome, thanks.
I have a few questions on restriction enzymes. Is there any determining factor on which enzyme you use for the forward and reverse primer? I often see protocols specifying that BamHI for instance be put on the forward primer...is this like a hard ands fast rule? Does it have anything to do with the position of the restriction sites on cloning vectors?

Edited by taiju, 17 April 2013 - 04:12 PM.


#17 bob1

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Posted 18 April 2013 - 02:17 PM

It mostly has to do with the position on the vector relative to the promoter site.

#18 taiju

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Posted 11 May 2013 - 06:46 AM

Just wanted to post a thank you to eveyone who replied to this topic and helped me out with a special shout out to bob1 :) I got an A on my paper and commended on my primer design so thank you so much.





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