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cleanup of digestion before ligation


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6 replies to this topic

#1 philman

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Posted 25 August 2010 - 06:30 AM

Hi,
I am ligating a gene which has had BclI restriction sites attached to either end via PCR into a BamHI site in a vector.

I am only using one enzyme on the insert and one enzyme on the vector, so there should be no other pieces of DNA in either reaction except for the 7-8bp that will be chopped off the ends of the insert when it is cut.

Since there are no other DNA pieces in either reaction mix, is it necessary to gel purify the DNA after digestion and before dephosphorylation of the ends of the vector, and again before I ligate the insert and vector together? and if it is neccecery to purify the DNA, is it possible to just use a QIAGEN PCR purification column instead of going abot the gel purification?

BamHI is heat-inactavated, so I can inactivate that before the phosphorylation step, at which point I can also use the heat-inactivated TSAP thermosensitive Alkaline Phosphatase to dephosphorylate the ends of the vector. So I don't see why I would have to purify the DNA if all the enzymes can just be inactivated.

Thanks for any replies, just hoping to be able to cut out a couple of steps!

#2 almost a doctor

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Posted 25 August 2010 - 07:32 AM

In my opinion, YES you do need to do gel extraction as you need to separate your uncut vector from the digested one. Doing so will save you having bigger problems in the downstream processes.
You could probably get away with just using a PCR clean kit for the digested PCR, but if you are running a gel for the vector you may as well run the PCR product in it.

Finally, I was under the impression that BamHI CAN NOT be heat inactivated, so you'll need to purify your diggested vector before dephosphorilation.

Just my thoughts, hope it helps.

#3 philman

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Posted 25 August 2010 - 07:47 AM

In my opinion, YES you do need to do gel extraction as you need to separate your uncut vector from the digested one. Doing so will save you having bigger problems in the downstream processes.
You could probably get away with just using a PCR clean kit for the digested PCR, but if you are running a gel for the vector you may as well run the PCR product in it.

Finally, I was under the impression that BamHI CAN NOT be heat inactivated, so you'll need to purify your diggested vector before dephosphorilation.

Just my thoughts, hope it helps.

The info leaflet that came with the BamHI says that it has 95% inactivation at 65 degrees for 15 mins, although the BclI info says that it is not heat inactivated. and why would an active RE interfere with a phosphatase anyway?

the point about seperating undigested from digested is fair enough though, although could I not just overdigest so that there is virtually no undigested left anyway?

Edited by philman, 25 August 2010 - 07:51 AM.


#4 perneseblue

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Posted 25 August 2010 - 08:25 PM


In my opinion, YES you do need to do gel extraction as you need to separate your uncut vector from the digested one. Doing so will save you having bigger problems in the downstream processes.
You could probably get away with just using a PCR clean kit for the digested PCR, but if you are running a gel for the vector you may as well run the PCR product in it.

Finally, I was under the impression that BamHI CAN NOT be heat inactivated, so you'll need to purify your diggested vector before dephosphorilation.

Just my thoughts, hope it helps.

The info leaflet that came with the BamHI says that it has 95% inactivation at 65 degrees for 15 mins, although the BclI info says that it is not heat inactivated. and why would an active RE interfere with a phosphatase anyway?

the point about seperating undigested from digested is fair enough though, although could I not just overdigest so that there is virtually no undigested left anyway?


The gel purification is also to remove any genomic DNA or polysaccharides contaminating the plasmid prep. The NEB website clearly indicates that BamHI can not be heat inactivated. (Thus I would gel purify the vector)

As for the PCR product, any nonspecific band produced in the PCR, will have been synthesized with the primer pair, and thus contain the BclI site.

Thus these contaminating DNA fragments will ligate to the vector, increasing the "background" of vectors carrying random junk. This problem can be overcome by screening more colonies. How many more colonies depends on how clean the PCR product and vector prep was. If the PCR product looks clean and the vector prep also looks clean, you can get away and avoid gel purification. But I personally gel purify every DNA fragment I work with. Do it right and do it only once.
May your PCR products be long, your protocols short and your boss on holiday

#5 mole

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Posted 25 August 2010 - 11:52 PM

HiDear,

I am totally agree GEL PURIFICATION IS NOT NECESSARY if you dont have two different fragment or a clean PCR product.

And as regarding to REstriction enzyme or other enzymes, There exist chaotropic agent like Gn-HCl in all kits whether it is PCR clean Up kit or Gel purification kit.

As i am just purifying products with PCR Cleanup kit and This exist in property of all columns that they wont bind DNA less than 20Bps.

As per my opinion , you can go ahead without gel purification if your release is just 7-8 bps only.

With Thanks and Regards,
Mole .

#6 philman

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Posted 26 August 2010 - 01:12 AM

I am using Promega enzymes, and strange that NEB BamHI cannot be heat inactivated but the Promega one can: http://www.promega.c...ing/REtable.pdf But I suppose that one is a recombined version or something

hmm I appreaciate the point about secondary products from the PCR also cutting with BclI and creating annoying religated vectors, I will gel purify before ligation then, but is it strictly necessary to do so to the vector between BamHI cutting and dephosphorylation of the vector ends, I don't see why BamHI present, even if it is active, would affect the alkaline phosphotase.

#7 HomeBrew

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Posted 26 August 2010 - 05:05 AM

It is not necessary to gel purify your vector digestion before dephosphorylation; in fact you can just add your alkaline phosphatase to your restriction digest mix for the last 10 minutes of the digestion.

The way we routinely proceed is to get our vector and insert digestions going, prepare a TAE gel with 1 mM guanosine added and let it solidify, add alkaline phosphatase to the vector digestion ten minutes before the two hour digestion is up, then at the two hour mark, load the samples immediately on to the gel submerged in TAE with 1 mM guanosine as running buffer, run the gel, and recover the fragments of both the vector and insert with a gel purification kit. There is no heat inactivation step needed.

We have found the gel purification step of both the vector and insert to be a necessary step -- trying to save time here by skipping this step more often than not results in having to repeat the experiment again due to failure to isolate the desired clone, thus not doing the gel purification is really a false economy in terms of saving time and reagents.




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