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Annealing Issues


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#1 Daccame

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Posted 15 June 2010 - 10:28 AM

I am having trouble with an annealing reaction where I need to clean the single stranded oligos from the tube containing the double stranded product as the oligos would interfere with the downstream parts of my protocol. Are there any suggestions as to how to do this?

I have tried running an agarose gel, which produces two bands. I have thought about cutting out the larger band, but this is complicated by the fact that single stranded DNA does not migrate in a predictable fashion on a gel.

Any advice would be appreciated.

#2 TomH

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Posted 16 June 2010 - 03:56 AM

Aren't PCR clean-up kits designed to remove small oligos?

#3 Micro

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Posted 16 June 2010 - 04:23 PM

Gel and PCR clean up kits are good for removing oligos from product that is >100bp or if there are multiple PCR product in a reaction.

However, if your product is <100bp or there is only one product in your reaction then using an combination of Exonuclease I and Shrimp Alkaline Phosphatase can be used to remove oligos. ExoSAP-IT is product by GE that does this but there are plenty of companies that have an equivalent product.




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