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Double Digest pCD Vector

Double Digest restriction enzyme

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#1 Vahe

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Posted 28 April 2015 - 10:59 AM

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.



#2 phage434

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Posted 28 April 2015 - 11:23 AM

Please give details of the reaction setup: volumes, reagents, heating, gel running. Do you have a gel image?



#3 Vahe

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Posted 28 April 2015 - 01:24 PM

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.



#4 phage434

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Posted 28 April 2015 - 01:31 PM

Do you get single cutting of your vector?  You can tell this by running a gel with uncut, cut with each, and cut with both enzymes. I would normally use a bit more enzyme, like 2 ul, but the reaction setup seems fine.  What is the volume of the DNA going into the reaction (not the amount)?  High volume of DNA solution can sometimes contain inhibitors, such as ethanol, which prevent the enzymes from working.  It is important the vast majority of the volume be water, not DNA containing solution for this reason. You should rarely need to cut this much DNA, so perhaps you should concentrate on cutting 100 ng instead.







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