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Need to add 12 bp at the beginning of the sequence


Best Answer phage434, 22 February 2016 - 06:29 PM

No, that won't work. Your primer has to anneal well at the 3' end, and this will not anneal at all at the 3' end. You'll need to make a primer

5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNN....3'

There should be no problem making this primer.

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#1 lexi1096

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Posted 22 February 2016 - 02:22 PM

I have DNA sequence to which I want to add 12 bp sequence starting at the position 16 bp. Is it possible to make this insertion using PCR? If I design primer that is 27 bp long (15bp matching the beginning of the sequence +12bp insert), do you think it will that work? Any other suggestions? My DNA sequence is 1200 bp long and the reverse primer will be designed appropriately.

 



#2 bob1

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Posted 22 February 2016 - 02:49 PM

Absolutely this will work by PCR. Don't worry about the length of the primers, just use the approximately 20bp that you would regularly use for a PCR, and add the additional bases to the 5' end of the primer. The additional bases do not count towards the annealing temperature of the PCR as they are non-specific, but will get incorporated after the first few rounds of the PCR cycle.



#3 lexi1096

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Posted 22 February 2016 - 04:22 PM

Absolutely this will work by PCR. Don't worry about the length of the primers, just use the approximately 20bp that you would regularly use for a PCR, and add the additional bases to the 5' end of the primer. The additional bases do not count towards the annealing temperature of the PCR as they are non-specific, but will get incorporated after the first few rounds of the PCR cycle.

I actually want the extra stuff to be inside of the sequence. Let's say I have a sequence 5' NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN....3' and want to insert XXXXXXXXXXXX with PCR, so that I have 5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNN....3'

 

So basically my question was how can I insert the sequence X with PCR? Will it work if my primer is 5' NNNNNNNNNNNNNNNXXXXXXXXXXXX?



#4 phage434

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Posted 22 February 2016 - 06:29 PM   Best Answer

No, that won't work. Your primer has to anneal well at the 3' end, and this will not anneal at all at the 3' end. You'll need to make a primer

5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNN....3'

There should be no problem making this primer.



#5 lexi1096

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Posted 08 March 2016 - 08:28 AM

Thanks everyone for their help! I used 5' NNNNNNNNNNNNNNNXXXXXXXXXXXXNNNNNNNNNNNNNNNNN primer and it worked, the 12 bp region was inserted in the right place :)






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