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need a method for oligonucleotide visualization


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5 replies to this topic

#1 Pazit

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Posted 27 November 2013 - 02:52 AM

Hi,

 

If I have an oligonucleotide of 22 bases, and the best readout for an assay I want to perform would be whether or not this oligonucleotide remains intact.

I can't run it on an agarose gel because it's too small, right? Is there a simple method I can use, or maybe a bioanalyzer? I know that I can run a PCR with this oligo as a primer and see if it works, but I need something faster and quantitative.

 

Thanks!



#2 phage434

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Posted 27 November 2013 - 06:56 AM

A 20% PAGE gel will resolve fragments of this size. Some HPLC systems will also be able to separate these. You could also do mass spec on the fragment.



#3 pcrman

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Posted 27 November 2013 - 07:59 AM

I am sure you can see a clear band on an agarose gel if it is intact. Just do it to see what you have. BTW, Is it RNA or DNA, single or double-stranded? 



#4 Pazit

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Posted 27 November 2013 - 10:19 AM

It's ssDNA. I'll probably try agarose, if it doesn't work I'll try PAGE. If that doesn't work then this readout is not suitable, since I need to run the assay on a daily basis so it doesn't make sense to use HPLC or mass spec. Thanks!



#5 phage434

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Posted 27 November 2013 - 10:25 AM

Why would you ever need to check an oligo every day? Freezers work, mostly, and it will be stable in TE at room temperature for months.



#6 Pazit

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Posted 27 November 2013 - 10:27 AM

It's part of the experiment. We are developing a method for detection of point mutations, and whether or not the oligo is intact can tell me whether or not there is a mutation.






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