sequantial digest - (Jun/18/2014 )
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?
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.
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?
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.
Ok
thanks for the answer bob1
I'll try that.
I am trying to remove a 200bp band from a 10.000 bp plasmid.
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.
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.
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.
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!
Here's a thread:
and a company offers column regeneration kits:
http://www.applichem.com/en/products/maxxbond/