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Annealed oligos vs. non annealed. Electrophoresis pattern.

oligo annealing shrna

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#1 Raquelina

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Posted 10 December 2012 - 10:47 AM

Hi everyone!

I'm cloning a shRNA sequence in plko.1 puro vector. I've already done the annealing step for the oligos, and now I'm trying to see whether they're annealed or not. I'm loading the same amount of either annealed or not annealed oligos and running a 0.8 agarose electrophoresis gel. My question is: Which oligos should run further? I'm getting a band at 25kb more or less for the non-annealed oligos, but I'm not able to see the annelaed ones.

Thanks a lot!

#2 bob1

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Posted 10 December 2012 - 12:12 PM

You get an oligo band of 25,000 bp? Wow, your oligos must be huge - at that sort of length how did you get them synthesized and purified enough to consider annealing (given that you should have HPLC purified for this sort of thing)?

Single stranded should run a bit faster I think, but the difference will be very slight.

#3 Raquelina

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Posted 10 December 2012 - 02:29 PM

Oops! I said 25kb, sorry, I meant 25 bp! I can only see ssDNA... Thanks Bob

#4 bob1

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Posted 10 December 2012 - 06:44 PM

To resolve 25 bp you need to run an acrylamide gel. Even 2% agarose will not resolve 25 bp well, let alone 0.8%.

The easiest way to check if your oligos are annealed would be to do the ligation into a vector and then amplify across the insert site with sequencing primers.

#5 Raquelina

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Posted 11 December 2012 - 12:17 AM

Thanks again! Really helpful!




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