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one question about TA cloning

one question about TA cloning - Molecular Cloning Forum

one question about TA cloning - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 11-28-2012, 10:35 PM
Pipette Filler
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Smile one question about TA cloning



hi all,

I have one question about TA cloing. My insert has restrict enzyme sites at both end (5' end BamH I and 3' end Xhol I), If my insert is in the reverse orientation, can it still be digested ?

thanks in advance
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Old 11-30-2012, 06:59 AM
Pipette Filler
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Default Re: one question about TA cloning

Hi,
Even tough the insert ligated in reverse orientation you will be able to digest with the mentioned enzymes. What ever is the orientation, your insert is between the two restriction sites know. If these mentioned enzymes are cutting in between the insert , then you have to calculate the expected size fragments considering both the orientations. Based on the result you can judge the orientation of insert.
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