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sequantial digest


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#1 bioke

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Posted 18 June 2014 - 02:27 AM

Hallo all,

 

I would like to make a digest with BspEI and XmaI , their buffers are not compatible, so I need to do a sequential digest.

 

Now can I just use XmaI in the specific buffer (50mM NaCl) and after this reaction is done just add some of the BspeI buffer (100mM potassium acetate)?

Or the other way around.

I read on the NEB website somewhere you can do a sequential restriction reaction using the lowest salt concentration first and than the highest one.

 

Or are they not compatible at all?

 

 

 



#2 bob1

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Posted 18 June 2014 - 01:07 PM

From their double digest table it seems that BspEI has 0 activity in buffer 4 and XmaI has 0 activity in buffer 3, so I would clean up between reactions.



#3 bioke

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Posted 18 June 2014 - 09:23 PM

I see.

 

So you would suggest a gel clean up than?

 

Would I not lose a lot of the DNA? I heard sometimes you lose almost 80% of the plasmid DNA?



#4 bob1

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Posted 19 June 2014 - 02:45 AM

No, just an ordinary PCR cleanup will do between digests - save the gel extraction for the final step if it produces two or more largeish (50 bp or more) fragments, as smaller than 50 bp will pass through a PCR cleanup kit column.

 

You could also just do a simple ethanol precipitation, but you can lose a lot of DNA this way if you don't have much or large fragments.



#5 bioke

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Posted 19 June 2014 - 03:30 AM

Ok

thanks for the answer bob1

I'll try that.

 

I am trying to remove a 200bp band from a 10.000 bp plasmid.



#6 bob1

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Posted 19 June 2014 - 01:19 PM

Ok, in that case just PCR clean between digests and do a gel extraction after the second digest.  The first digest should give you only one band as it is a single digest.



#7 phage434

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Posted 19 June 2014 - 04:22 PM

I now almost universally use ampure bead cleanup for PCR and digest reactions, which I find efficient and much easier, especially with many samples and a multichannel pipettor. You probably won't see a 200 bp fragment digested from a 10 Kb plasmid on a gel unless you load a LOT of cut plasmid, since the band intensity depends on mass, not molarity.



#8 Bio-Lad

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Posted 20 June 2014 - 04:54 AM

For what it's worth, you can do a simple column purification on your first digest and then digest the purified plasmid with your second enzyme.  We regenerate our miniprep columns and make our own gel-solubilization/DNA binding buffer so the cost is negligible over the long term and the results are always dandy!  I'd be happy to share the recipe and protocol we use.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#9 paulO

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Posted 20 June 2014 - 08:47 AM

Regenerating mini-prep columns sounds terrific.  I used to do TELT minipreps ( no columns needed ) and am a little embarrassed at having succumbed to the column craze.

 

Please share the protocols!



#10 hobglobin

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Posted 20 June 2014 - 10:19 AM

Here's a thread:

http://www.protocol-...tion#entry19532

and a company offers column regeneration kits:

http://www.applichem...ducts/maxxbond/


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#11 bioke

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Posted 21 June 2014 - 12:08 AM

Ok, in that case just PCR clean between digests and do a gel extraction after the second digest.  The first digest should give you only one band as it is a single digest.

 

The first digest: it gave 2 bands! Very close next to eachother. Is this uncut DNA vs Cut DNA? I suppose the higher band is the cut DNA?

 

 

@phage434: Indeed, I will hardly see it if the band is removed or not. this brings me to the my other question. How can I know its the right plasmid I will have to transform the cells with?

There is 1 restriction site in the part I remove, but not sure this will help to see if I have the correct plasmid. Or would I simple see it as uncut plasmid vs cut plasmid on a gel? (as I saw something similar now from the first digest).

 


 



#12 bob1

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Posted 21 June 2014 - 02:38 AM

 

Ok, in that case just PCR clean between digests and do a gel extraction after the second digest.  The first digest should give you only one band as it is a single digest.

 

The first digest: it gave 2 bands! Very close next to eachother. Is this uncut DNA vs Cut DNA? I suppose the higher band is the cut DNA?

Maybe - did you run a control of uncut DNA on the gel?  That should show you if the band ispartially uncut or not.  Another option is that there is more than one restriction site in the plasmid.



#13 bioke

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Posted 21 June 2014 - 05:10 AM

 

 

Ok, in that case just PCR clean between digests and do a gel extraction after the second digest.  The first digest should give you only one band as it is a single digest.

 

The first digest: it gave 2 bands! Very close next to eachother. Is this uncut DNA vs Cut DNA? I suppose the higher band is the cut DNA?

Maybe - did you run a control of uncut DNA on the gel?  That should show you if the band ispartially uncut or not.  Another option is that there is more than one restriction site in the plasmid.

 

 

Yes!

And thats the weird thing:

 

The control DNA was (at least this is how it looked) located "between" the cut sample!

(but the control sample was very thick..., so not too clear, I also could not take a picture, will need to do it next time).

 

And no: there is normally only 1 of that restriction site (according to the sequence I have at least)

 



#14 bioke

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Posted 23 June 2014 - 09:38 AM

No, just an ordinary PCR cleanup will do between digests - save the gel extraction for the final step if it produces two or more largeish (50 bp or more) fragments, as smaller than 50 bp will pass through a PCR cleanup kit column.

 

You could also just do a simple ethanol precipitation, but you can lose a lot of DNA this way if you don't have much or large fragments.

 

Just curious, when you do the PCR cleanup in between: do you bother to heat inactivate the enzyme? I think its not needed because you will "lose" the enzyme during the clean up (will go through the column).

Or is this incorrect?

 



#15 phage434

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Posted 23 June 2014 - 09:41 AM

You don't really care here. If the enzyme is active during the second digestion, so what? The only difficulty would be if it showed star activity (unlikely). You do need to disable/eliminate the enzyme prior to ligation, assuming you are recreating the cut site. If not, you can do a simultaneous cut/ligate reaction, cycling betwen 37 and 16 if necessary.






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