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Purification for TOPO


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#1 Biologystudent

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Posted 04 April 2014 - 05:11 AM

Which approach is the best for purification of a sample for TOPO?

 

a) run a small PCR-sample on electrophoresis (5ul). If I get a clear band I will then purify the other PCR-sample (45ul) that I did not run on agaros electrophoresis

 

b ) run 2 lanes of electrophoresis, one lane with 5ul and one with 45ul. The one with 5ul I expose to UV to see whether I get a clear band, and if I do then I purify the PCR-product from the other agaros-electrophoresis (which I save from UV-exposure).

 

I am thinking that a) is a more preferable approach since purification from a solution would give BETTER YIELD than purification from agaros? Any comments on this?



#2 manziki

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Posted 04 April 2014 - 01:36 PM

They can both work perfectly but it depends first on the yield of your PCR and second on the specificity of your primers.

In case you get a single band, use the second approach, If you get multiple bands cut out the desired one from the gel and clone it (here you can mix the identical bands to increase the yield)






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