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BamHI restriction digest


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9 replies to this topic

#1 plasmodel

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Posted 17 June 2009 - 06:38 PM

Hi,

I just did a plasmid prep and went on to do a single restriction enzyme digest with BamHI. After cleaning it up, I found that there were three bands on a gel (one is undigested; one is digested) but the last one? I guess you could say star activity but is there any other way for me to check where I might have screwed up? I speced my plasmid prep as well as the restriction digested sample and both were relatively pure (260/280 was around .8).

I ran the same reaction several months ago and it was fine (2 hrs 37C). What should I do?

#2 molgen

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Posted 18 June 2009 - 12:52 AM

It can also bee uncut supercoiled plasmid

#3 eldon

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Posted 18 June 2009 - 10:01 AM

It can also bee uncut supercoiled plasmid


bamh1i is notorious for star activity...especially depending on the source of the enzyme

always run uncut control lanes so you can distinguish your supercoiled band from star activity...and use minimal enzyme. you can always do mock or prelim digests to determine optimal cleavage (amt. of DNA: RE) prior to gel extraction

Edited by eldon, 18 June 2009 - 10:02 AM.


#4 HomeBrew

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Posted 18 June 2009 - 06:15 PM

BamHI is the most frequently used enzyme in my lab, and we've never seen star activity. Are you sure the digest worked at all? If it didn't, you're likely seeing three forms of the plasmid DNA. Do as eldon suggests -- run out an undigested control.

#5 jiajia1987

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Posted 18 June 2009 - 07:01 PM

It is good to do what HomeBrew has suggested.

I get different results when I digest my plasmids or inserts with BamH1 for different durations.

#6 plasmodel

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Posted 23 June 2009 - 05:53 PM

I redid the restriction digest with a new plasmid prep. And, I see the band that I want but there is also an additional band (bigger). I also ran a RD using another plasmid prep but the same thing happened again. Yes, I ran the pp as well.

I don't know what to do! Can I do a TAE gel and just remove the band and try recover the DNA from there? Does anyone have a good protocol for that?

#7 HomeBrew

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Posted 23 June 2009 - 06:46 PM

Maybe you're leaving your plasmid prep in the alkaline lysis step too long? This can irreversibly denature the DNA... Or there's something wrong with the enzyme -- maybe it's expired?

In any event, if you're getting partial cutting and there's a linear band of appropriate size being liberated, you can cut this out of a gel and proceed.

#8 plasmodel

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Posted 24 June 2009 - 08:52 PM

Maybe you're leaving your plasmid prep in the alkaline lysis step too long? This can irreversibly denature the DNA... Or there's something wrong with the enzyme -- maybe it's expired?

In any event, if you're getting partial cutting and there's a linear band of appropriate size being liberated, you can cut this out of a gel and proceed.



This simple restriction digest step is dragging on for too long------I did the TAE gel and one of the samples was fine but what do I see for the other, a huge band (that looked spikey, not a pretty band)....It must probably have been because it had a really high concentration (>400ng/ul). So, I am rerunning the RD and will redo the TAE. Does anyone have any idea as to why this might be the case?

#9 swanny

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Posted 25 June 2009 - 06:35 PM

A couple of questions:
What were the sizes of the three bands after digestion? I would not suspect star activity unless you hadd small bands or a smear.
We have found that cells grown in LB might give lower plasmid yields, but the DNA is always of higher quality for digestion than if we grow the cells in 2YT.
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#10 phage434

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Posted 28 June 2009 - 12:00 PM

This is just a guess, but from what you have said I get the impression that you are trying to digest too much DNA in this reaction. More is not better. Aim for 1 ug of DNA in a 50 ul restriction digest (or scale up the reaction to cut more DNA). Perhaps you could tell us precisely the reaction conditions you are using.




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