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Blunting both vector and insert


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#1 calvin*

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Posted 09 March 2005 - 06:39 AM

Hi friends,
am encountering problems with blunting the vector (EcoRI site) and insert ( EcoRI and SalI ends) using ROCHE Klenow. After I carryout the bluting reaction (for both)and CIP for the vector, I am losing DNA completely. Is Problem with enzyme? or water or many gel extractions? Is their any way in reducing DNA gel extraction steps?(I use freeze thaw extraction)?Please help as it is taking a lot of valuable time.
Thanks
"In my opinion, we don't devote nearly enough scientific research to finding a cure for jerks"-Calvin

#2 mearegp

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Posted 09 March 2005 - 08:05 PM

You might try T4 polymerase instead of Klenow. This will fill in the overhangs to give blunt ends. As for gel extractions, they are not necessary ( although you probably need to gel extract your insert to start with), after the digestion reaction you can heat inactivate the enzymes, isopropanol precipitate, wash with 70% ethanol, and resuspend in water. Or if you have a PCR clean up kit you can use it to recover your DNA. Hope this is helpful.

Good Luck

#3 fred_33

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Posted 10 March 2005 - 12:34 AM

hi
i suppose that the better way to extract blunted and "CIPed" fragments is a precipitation with salts and ethanol. A gel extraction reduces strongly the efficiency of recovery.

Fred




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