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Restriction digest


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7 replies to this topic

#1 roxanne911

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Posted 22 October 2009 - 06:24 AM

Hi, I am new to do research. I found that normally the recognition sequence of restriction enzyme is from 5' to 3', for example, the recognition site for BamHI is

5' GGATCC
3' CCTAGG

If the sequence of my gene is

5' CCTAGG
3' GGATCC

Can I use BamHI to cut my DNA?

#2 Warren

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Posted 22 October 2009 - 08:11 AM

Hi, I am new to do research. I found that normally the recognition sequence of restriction enzyme is from 5' to 3', for example, the recognition site for BamHI is

5' GGATCC
3' CCTAGG

If the sequence of my gene is

5' CCTAGG
3' GGATCC

Can I use BamHI to cut my DNA?


No.

Warren..

#3 swanny

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Posted 25 October 2009 - 05:40 PM

Roxanne, go to the NEB website (http://www.neb.com/n...nzymeFinder.asp) and put in different sequences.
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#4 Julio-Claudian

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Posted 26 October 2009 - 12:20 AM

Dear all,

If I may piggyback on this thread to avoid opening a new one..

I've been following the info sheet that comes with the RE (e.g. 1 hour at X 'C) but BamHI doesn't give anything. The plasmid's still intact. Veterans doing restriction digest suggest doing it overnight and as much as I wanted to follow, the sheet says extended incubation not more than 8 hours.

Tips?

Thanks.

#5 Prep!

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Posted 26 October 2009 - 01:49 AM

are you using the buffer system that the sheets says too??!!
you can do it overnight and follow it once. It might lead to star activity sometimes but as you say there is no cut in 8 hours, you can give it a try!!!
r try increasing the Enzyme concentration or decreasing the sample conc.
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#6 phage434

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Posted 26 October 2009 - 04:04 AM

Is this plasmid DNA that has been cloned in E. coli? Genomic DNA or plasmids from other species can contain modifications that will inhibit cutting.

What is your DNA concentration? Often cutting too much will make it look as if nothing is happening.

What volume are you cutting in? Too much enzyme in a reaction will inhibit reactions.

Are you sure the plasmid has these cut sites?

#7 Julio-Claudian

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Posted 27 October 2009 - 06:21 AM

Thanks Pradeep and phage434. Yes, used the correct buffer. I've done an overnight (< 16 hours) digestion before I got back a while ago to see how things go.

Am digesting 400 ng (2 x 200 ng/uL) of my DNA using total of 10U of enzyme. And it seems to be a norm for them whom I've spoken to; to do an overnight digest at 37'C. By that "norm" I meant to say that it's 37 regardless of the enzyme

e.g. BstBI at 37 (although NEB recommends 65) and get what they desired..

Edit: Perhaps lowering the amount of enzyme for prolonged incubation, no? :)

Thanks

Edited by dreamchaser_jc, 27 October 2009 - 06:32 AM.


#8 arnabde2000

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Posted 27 October 2009 - 09:03 AM

If the restriction site for BamH1 is
5' GGATCC
3' CCTAGG,
it will not cut
5' CCTAGG
3' GGATCC

that is what I think. Am I correct? What does your result show?




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