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Smallest insert in PCR

PCR

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6 replies to this topic

#1 rameshcdri

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Posted 10 January 2012 - 11:32 AM

Hi All,

I am doing small gene insert in my gene of interest (450bp). My insert are very small length 6bp and 9 bp.

Is this insert could distinguish in Agarose gel when compare with non insert PCR product?

Thanks.

Ram

#2 bob1

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Posted 10 January 2012 - 12:49 PM

No. You might (should) be able to distinguish them using sequencing acrylamide gels and/or sequencing

Edited by bob1, 10 January 2012 - 12:50 PM.


#3 Trof

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Posted 11 January 2012 - 07:20 AM

Or fragmentation analysis (usually the same machine as sequencer). That would be most suitable IMO, since it can even quantify them. You would need one of your regular PCR primers to be re-synthethised with a fluorescent dye though.

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#4 phage434

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Posted 11 January 2012 - 08:28 AM

You can pcr amplify only a small portion of your gene, surrounding the insertion. This will give fragments with a larger relative difference in length. Then, running these on a 20% TBE gel, you should be easily able to determine the insert presence compared to a gene without the insert.

#5 rameshcdri

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Posted 21 January 2012 - 07:00 PM

Thanks to all.

#6 scolix

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Posted 30 January 2012 - 09:04 AM

to be absolutely sure, i will sequence it.

#7 rameshcdri

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Posted 17 February 2012 - 01:13 PM

Thanks.





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