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Pcr primers

Cloning question

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7 replies to this topic

#1 jackster101

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Posted 06 February 2014 - 07:40 PM

I'm designing primers to simplify a full length cDNA. My issue is the small number of complementary bases!

Forward primer contains 6 extra bases plus restriction site plus Kozak plus 7 complementary bases! This seems to not sound right??!

#2 Ameya P

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Posted 06 February 2014 - 09:56 PM

Dear Jackster, 

 

I suppose you should have 15-18 bases complementary to ensure that you are amplifying the region you want. 


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#3 perneseblue

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Posted 07 February 2014 - 05:20 PM

I agree with Ameya P. You do need more bases.


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#4 jackster101

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Posted 10 February 2014 - 07:05 AM

Thanks!
I guess I'll have to do multiple pcr rxns to achieve my goal. My one concern is that I'm amplifying a 7kb cDNA and feel that 3 pcr rxns will increase probability of mutations. Guess I'll need to sequence a lot of clones!

#5 phage434

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Posted 10 February 2014 - 08:39 AM

I am completely mystified by  your comment. Your primers should like like this:

5' [six bases of trash DNA] [restriction site] [Extras such as Kozak] [18-22 bp matching your target] 3'

What does this have to do with how many PCR reactions you need?



#6 jackster101

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Posted 13 February 2014 - 07:14 AM

Given the restriction on the Tm being between 65-75 degrees, I can't make my primers very long.- right ? My primers were close to 70 degrees. Btw- my kozak seq was 6 BP.

I have 6 BP extra seq plus 6 BP restriction site plus 6 BP Kozak

#7 phage434

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Posted 13 February 2014 - 07:25 AM

You can make your primers as long as you would like. Don't pay attention to the Tm reported by the synthesis companies. The Tm of the 18-22 bases matching your sequence is what matters most. So, the reported Tm will be very much higher than the effective Tm for your PCR reaction.



#8 jackster101

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Posted 16 February 2014 - 04:25 PM

Thanks guys! Appreciate it! I'll order the new set today.,




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