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How to clean up restriction enzyme digestion


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#1 NatiCarol

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Posted 24 April 2009 - 01:12 PM

Hi
I´m digesting a pET28a plasmid with NdeI and HindIII . Although there is buffer compatibility, I could not set up a full digestion in the same buffer (I have tried many buffers).
So, between NdeI and HindIII digestion I have to clean up the mixture.
I was wondering if I can use a Sephacryl 200 spin column or if I can use a GFX column, like I use to clean up PCR reactions. I use both of this columns to clean PCR products.
I would like to know if anyone use this columns or if just gel-purified plasmids can be used for sequential digestion.
I would like to know also if, after I include SAP (phosphatase), I have to gel-purify or if I can just do a purification in S-200 or GFX.
Thanks
Natássia

#2 T C

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Posted 24 April 2009 - 09:00 PM

Hey,

Pick what you like, I would prefer double digestions as it saves time and efforts.

!. I prefer using a clone where some fragment is already inserted in these sites as then I can easily distinguish between double cut and single cut vector.

2. SInce you cannot distinguish between the single and double cut vector I assume that you figure out that its incomplete digestion on the basis of religations of the vector that you get. In this case you can CIP the digests.

3. If your restriction enzymes are working fine and you still get incomplete digestions, use less of DNA for digestions and setup multiple digestions, which you pool during gel elutions.

4. If you still wana go for sequential digestions, just precipitate the DNA after the first digestions with ammonium acetate and ethanol. You can also use a column but it proves out to be more expensive.

Hope it helps.

Best,
TC

Hi
I´m digesting a pET28a plasmid with NdeI and HindIII . Although there is buffer compatibility, I could not set up a full digestion in the same buffer (I have tried many buffers).
So, between NdeI and HindIII digestion I have to clean up the mixture.
I was wondering if I can use a Sephacryl 200 spin column or if I can use a GFX column, like I use to clean up PCR reactions. I use both of this columns to clean PCR products.
I would like to know if anyone use this columns or if just gel-purified plasmids can be used for sequential digestion.
I would like to know also if, after I include SAP (phosphatase), I have to gel-purify or if I can just do a purification in S-200 or GFX.
Thanks
Natássia



#3 mastermi

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Posted 24 April 2009 - 10:58 PM

4. If you still wana go for sequential digestions, just precipitate the DNA after the first digestions with ammonium acetate and ethanol. You can also use a column but it proves out to be more expensive.


Yes, ethanol-acetate precipitation would be the best way if you just want to change the buffer conditions. Not only because it's less expansive, but because you loose less DNA compared to a spin column.

#4 NatiCarol

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Posted 25 April 2009 - 09:44 AM

4. If you still wana go for sequential digestions, just precipitate the DNA after the first digestions with ammonium acetate and ethanol. You can also use a column but it proves out to be more expensive.


Yes, ethanol-acetate precipitation would be the best way if you just want to change the buffer conditions. Not only because it's less expansive, but because you loose less DNA compared to a spin column.



Thank you very much... But in this ethanol-acetate precipitation do I have to do a phenol extraction first or I simply put ethanol and acetate and precipitate at - 20C? Thanks

#5 T C

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Posted 25 April 2009 - 10:12 AM

Hey,

Just add 1 volume NaOAc, 3M, pH 5.2 to the digests and 6 volumes ethanol, keep for 2 hrs at -20, spin, wash with 70 % ethanol, dry and setup the second digestion.

Best,
TC

Thank you very much... But in this ethanol-acetate precipitation do I have to do a phenol extraction first or I simply put ethanol and acetate and precipitate at - 20C? Thanks



#6 NatiCarol

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Posted 25 April 2009 - 10:20 AM

Hey,

Just add 1 volume NaOAc, 3M, pH 5.2 to the digests and 6 volumes ethanol, keep for 2 hrs at -20, spin, wash with 70 % ethanol, dry and setup the second digestion.

Best,
TC

Thank you very much... But in this ethanol-acetate precipitation do I have to do a phenol extraction first or I simply put ethanol and acetate and precipitate at - 20C? Thanks


thanks TC!!

#7 hanming86

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Posted 01 May 2009 - 06:30 AM

remember to heat inactivate at the end of the digestion. I think HindIII is heat labile. avoid phenol chloroform whenever possible . residual phenol is a killer to most enzymatic application.
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#8 perneseblue

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Posted 01 May 2009 - 06:52 AM

If you ethanol precipitate and wash your DNA (with 70% ethanol) after the phenol extraction step, any phenol residue would be removed.
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#9 scolix

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Posted 01 May 2009 - 11:35 PM

I would heat inactivate it and do an ethanol precipitation. But for these enzymes, ethanol precipitation is sufficient.




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