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Vahe's Content

There have been 2 items by Vahe (Search limited from 22-November 18)


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#174606 Double Digest pCD Vector

Posted by Vahe on 28 April 2015 - 01:24 PM in Molecular Biology

I'm running a 50 uL reaction with 5uL of 10x buffer (either Cutsmart or 2.1), 1uL of each enzyme and 1ug of DNA although I've also gone up as high as 2ug of DNA. Heated to

37 degrees C. I don't have a gel image but there was definitely nothing that popped out. All I had was a band for my vector  but nothing else.




#174602 Double Digest pCD Vector

Posted by Vahe on 28 April 2015 - 10:59 AM in Molecular Biology

Hi everyone,

 

I have been battlin with two digestion reactions and could use some help. I'm trying to cut two pCD vectors that is 6525 base pairs long.

Reaction 1: Xho1/BbvC1

Reaction 2: Xho1/Xba1

 

I'm using enzymes from NEB. I've tried using both Cutsmart buffer and 2.1 buffer, both of which are compatible with these enzymes. I've tried running the reaction for 1 hour at 37oC and have even gone up to doing an overnight digestion. My cut out are 350 and 480 base pairs, respectively. I've cut up to 2 ug of DNA and still don't see a cut out.

 

If anyone had a similar problem with these or can provide any expertise in using these enzymes, I would appreciate the insight.  Thanks.





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