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doubt in cloning

doubt in cloning - Molecular Biology Techniques

doubt in cloning - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 04-10-2012, 04:19 PM
Pipette Filler
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Default doubt in cloning



Hi,

i hav a Pcr product for site EcoRI and Sma I but it has got by using taq polymerase (EcoRI-cohisive site) and Pfu polymerase (Sma I-blunt site) then digest Pcr product with EcoRI and gel purified the product was ligated with vector sequencially digested with Sma I and EcoRI gel purified with good DNA concentration, however after ligation could not get any transformed colonies,

i hav this doubt that, while using Pfu tag would it removes EcoRI site of PCR product so that the ligation fails are any other incompactibility may there,

how can i do clone the EcoRI / Sma I pcr product in vector digested with Sma I and EcoRI ?

Last edited by cloningproblem; 04-10-2012 at 04:20 PM.
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Old 04-11-2012, 09:55 AM
Pipette Filler
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