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Addition of Restriction sites into PCR primers


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4 replies to this topic

#1 ropa

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Posted 06 April 2010 - 03:00 PM

Hello,

I am new to cloning. I am trying to move my insert from a non expression vector to an expression vector. Because none of the restriction sites in the two vectors match, I have to design restriction sites present in the expression vector within my PCR primers so as to amplify the insert and then use the restriction sites to cut and then insert my insert. However, i was wondering if anyone has a step by step protocol on how to add restriction sites into PCR primers.

Thanks.

#2 phage434

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Posted 06 April 2010 - 03:46 PM

1. design normal primers

2. add the RE site to the 5' end
3. add 6 bp of random sequence to the 5' end (possibly more for extremely fussy enzymes)
4. check primers for primer-dimer and hairpins and self-priming (idtdna.com tools site)
5. redesign if necessary

In use, make sure to purify the pcr product before cutting with the enzyme(s). Cloning with different enzymes at each end is easier and yields directional control over the product.

#3 ropa

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Posted 06 April 2010 - 05:45 PM

1. design normal primers

2. add the RE site to the 5' end
3. add 6 bp of random sequence to the 5' end (possibly more for extremely fussy enzymes)
4. check primers for primer-dimer and hairpins and self-priming (idtdna.com tools site)
5. redesign if necessary

In use, make sure to purify the pcr product before cutting with the enzyme(s). Cloning with different enzymes at each end is easier and yields directional control over the product.


Thanks. I think I got all that. I guess I ahould ask another question. I have seen that in some protocols they ignore the stop codon when designing primers and others do not. Anyone know the reason for this because I am confused as to why they would exclude the stop codon in the design of the primers.

Edited by ropa, 06 April 2010 - 06:17 PM.


#4 pcrman

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Posted 06 April 2010 - 07:05 PM

Hi Ropa,

welcome to the forum.

Regarding adding extra bases to your oligos, you can visit this page http://www.protocol-...osts/36877.html

As for stop codon in insert, some vectors may already contain the stop codon downstream of the insert and there is no need to include it in insert.

#5 tea-test

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Posted 06 April 2010 - 10:49 PM

also, if you want to add a fusion tag like GFP downstream to your GOI you have to remove the stop codon.
tea-test: The artist formerly known as Ned Land




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