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for T4 poly filling reaction


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#1 bluetutu2010

bluetutu2010

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Posted 11 December 2010 - 10:13 AM

Hi guys,

I just found this nice forum. I have a question for T4 polymerase filling reaction. I have a plasmid on which I want to remove a fragment. The way to remove this fragment is to digest my plasmid by only two enzymes (eg. KnpI and EcoRI). My supervisor advived me doing filling reaction to delete the fragment. T4 poly filling reaction will fill the 5' termini and degrad 3' termini and then repaire the complete plasmid... I didn't purified the production... My supervisor also suggested to dilute the mix solution and then to inactivate the enzyme by heating (70C for 10min)...But I did not get what I want...the fragment was still there...It seemed that T4 recovered the original plasmid..Could anybody give me some advice? Maybe I should purified the production before the ligation...I will try again tonight... Also is there the protocol for filling reaction? I cannot find it from internet...

Have nice weekends for every one^^

Sincerely,




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