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Partial digest XhoI NotI NEB

Partial digest XhoI NotI NEB

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6 replies to this topic

#1 paakoo92

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Posted 29 May 2014 - 01:19 PM

Hi There.

 

I have a problem with the restriction enzyme XhoI and NotI form NEB.

I'm traying to cut 1ug of DNA (purified by Pure yield Midiprep system Kit from Promega) but I see partial digestion in the agarose gel.

I've tried to digest since 500 ng to 5 ug but the results are the same. I used low concentration enzyme (20 U/ul) to digest 500 ng and 4 ug and high concentration enzyme (100 U/ul) to digest 500 ng 1 ug 2 ug  and 5 ug. And I don't know wat I'm doing wrong.

 

I'm using the next conditions:

 

5 ul Buffer NEB2

0.5 ul BSA (100X)

38 ul DNA (130 ng/ul)

4.5 ul Water

1 ul XhoI (100 U/ ul)

1 ul NotI-HF (100 U/ul)

 

Incubation at 37 °C for 2 hours.

Desactivation 65 °C for 15 min.



#2 bob1

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Posted 29 May 2014 - 05:45 PM

have you tried sequential digestion?  Or tried individual digests to see if one of the REs isn't working?

 

Try using less enzyme - you should only need about 4-10x the amount of DNA (e.g. for 500 ng you would use 2-5 U).



#3 labtastic

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Posted 03 June 2014 - 05:26 AM

Use NEB buffer 3 for NotI and XhoI double digest. With buffer 2 NotI only has 50% activity (unless you are working with the high fidelity version of NotI in which case buffer 2 or 4 should work).

 

I just did this exact double digest a couple weeks ago with relatively old enzymes from NEB (not the HF versions) and it worked great with buffer 3. I also don't commonly use BSA either unless it is absolutely essential. 


Edited by labtastic, 03 June 2014 - 05:31 AM.


#4 Bio-Lad

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Posted 20 June 2014 - 04:47 AM

Labtastic,

He's Using NotI-HF, which is different in it's buffer preference from NotI (regular version).  

 

Paakoo,

The NEB app on my phone recommends using CutSmart buffer (which I believe is the same as Buffer 4 + BSA) for this digest.  Perhaps try that?  Definitely do as Bob suggested, though.  Do single digest controls (in the same buffer as you'd use for the multiple digest).  You may have dead or dying enzyme in one of your tubes and this could help you to know which it is.  I've had that happen before.

 

 

*Edited, Ooops I just saw the date on this post.  Odds are you've solved this by now =P 


Edited by Bio-Lad, 20 June 2014 - 04:48 AM.

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#5 phage434

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Posted 20 June 2014 - 05:23 AM

I think your problem is the high volume of DNA prep in your reaction. Almost all the volume is prepared DNA. If it has any impurities, then enzyme inhibition is likely. Common problems are ethanol contamination and Gu-HCl contamination. Try the digestion at higher dilutions, or purify your DNA again, making sure that the ethanol is really gone (high speed dry spin if using a column).



#6 Trof

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Posted 20 June 2014 - 07:21 AM

phage434: He wrote he tried concentrations from 500 ng to 5 ug, the posted protocol is for 5 ug. Chance is, in the 500ng one he had only around 3 ul of DNA and the rest was water. In that case the incomplete cut is unlikely to be caused by impurities in both of these cases.


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#7 phage434

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Posted 20 June 2014 - 07:38 AM

Good point.







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