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Dephosphorylation


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7 replies to this topic

#1 lucilius

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Posted 15 March 2013 - 11:56 AM

Hallo all,

I am wondering whether dephosphorylation is really needed to prevent selfligation.

Many protocols say you need to to do this to prevent self ligation, but how can a vector that has been cut selfligate without ligase present?

I am assuming if you do the digestion of the vector that you do not add ligase ... so how does the selfligation happen?

#2 macrox

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Posted 15 March 2013 - 12:59 PM

It does not "self ligate" without ligase, however it can "self ligate" during ligation reaction where ligase is present in the mixture. It is advisable to dephosphorylate digested plasmid vector before ligation to lower the number of colonies with circularized empty plasmid after transformation of the ligation mixture.

#3 bob1

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Posted 15 March 2013 - 04:51 PM

Only in the case of single or blunt cut vector - if you double cut with non-compatible ends, you shouldn't need to dephosphorylate.

Also note that the alkaline phosphatases used for this process tend to be very very stable, so despite the protocols saying that you can heat inactivate, this is often not the case.

#4 lucilius

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Posted 16 March 2013 - 01:39 AM

Only in the case of single or blunt cut vector - if you double cut with non-compatible ends, you shouldn't need to dephosphorylate.

Also note that the alkaline phosphatases used for this process tend to be very very stable, so despite the protocols saying that you can heat inactivate, this is often not the case.

Its indeed a single cut , just 1 RE that opens the vector.
But still: you do agree with the fact that it can not self ligate without the ligase?

And about the heat inactivate not working , you mean that if you do it you risk that the enzyme stil stays active while you are doing the ligase reaction with the insert?

#5 bob1

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Posted 16 March 2013 - 12:46 PM

For a single cut, I would dephosphorylate, but you want to use as little of the phosphatase as possible, otherwise you will often find that you will get no clones. CIAP is probably the worst of the phosphatases, though shrimp is pretty bad too, if you use one of the more recent phosphatases from cold organisms, then they tend to be less heat stable and hence more inactivatable.

The vector can not self ligate with ligase, but it is entirely possible that not all of the vector will cut with the RE in the first place.

#6 lucilius

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Posted 16 March 2013 - 01:23 PM

For a single cut, I would dephosphorylate, but you want to use as little of the phosphatase as possible, otherwise you will often find that you will get no clones. CIAP is probably the worst of the phosphatases, though shrimp is pretty bad too, if you use one of the more recent phosphatases from cold organisms, then they tend to be less heat stable and hence more inactivatable.

The vector can not self ligate with ligase, but it is entirely possible that not all of the vector will cut with the RE in the first place.

Ok thanks, but you do mean that the vector can not self ligate without ligase ?

Or?

#7 bob1

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Posted 17 March 2013 - 12:42 AM

Sorry, yes, typo - shouldn't religate without ligase.

#8 lucilius

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Posted 17 March 2013 - 01:40 AM

Sorry, yes, typo - shouldn't religate without ligase.


Ok thanks




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