Ligation - dephosphorylation?
KLADE Torsten
To all netters!
Is it absolutely necessary to dephosphorylate the target vector
when ligating single digested inserts?
I kept dephosphorylation with additional Phenol-Chloroform extraction,
but the efficience of the ligation didn`t seem to improve!
Any sugestions?
Thank you
Torsten
----------------------
Torsten Klade tor...@dgen10.gen.sbg.ac.at
University of Salzburg
Brian Foley
: To all netters!
: Is it absolutely necessary to dephosphorylate the target vector
: when ligating single digested inserts?
No. Not _absolutely_.
: I kept dephosphorylation with additional Phenol-Chloroform extraction,
: but the efficience of the ligation didn`t seem to improve!
: Any sugestions?
What do you mean by efficiency? You should get fewer Amp resistant
colonies, but a higher percentage to the colonies should contain
your insert.
If you are confident of something like a blue-white screen to tell you
which colonies have an insert, then dephosphorylation is not absolutely
needed. However, remember that without it, the vector can recircularize
and this will happen at a high rate unless you have a high concentration
of DNA. With a high concentration of DNA ends you will get less self-
circles, but you will start to get concatomers.
Read up on what the Molecular Cloning manual (Sambrook Fritsch and Maniatis)
pages 1.63 - 1.67 says about the j/i ratios. The original data was
published in:
Dugaiczyk,A. et al. (1975) Ligation of EcoRI endonuclease-
generated DNA fragments into linear and circular structures. J. Mol.
Biol. 96, 171
: Thank you
: Torsten
No problem.
--
********************************************************************
* Brian Foley * If we knew what we were doing *
* Molecular Genetics Dept. * it wouldn't be called research *
* University of Vermont * *
********************************************************************
Hinayana Bawagan
>To all netters!
>Thank you
> Torsten
I assume that the following is the sequence following up to
the ligation step:
1. linearize the vector
2. dephosphorylate the vector (may go directly from the digestion
thus omitting the phenol/CHCl3 extraction and Etol pptn steps)
3. do phenol/CHCl3 and Etol pptn steps and run DNA in a
low melting point agarose gel
4. extract DNA w/ any of the ff: the traditional methods as
described in Maniatis or by geneclean or by Boehringer Mannheim's
agarase
5. the insert of course should also be gel-purified after
restriction enzyme digestion
6. do the ligation in minimal volume (10 ul) with a large excess
of insert over dephosphorylated vector; also an excess of ligase
wouldn't hurt; have two controls: a) dephosphorylated vector alone
and NO LIGASE to check for carryover of unlinearized vector;
b) dephosphorylated vector alone WITH LIGASE to check for
the efficiency of dephosphorylation; ligation can be carried out
overnight at 12 C, 3-4 hrs at room temp and my labmates got it to
work also at 37 C 1 hr.
Duke Groebe
(KLADE Torsten) wrote:
>
>
> To all netters!
>
> Is it absolutely necessary to dephosphorylate the target vector
> when ligating single digested inserts?
> I kept dephosphorylation with additional Phenol-Chloroform extraction,
> but the efficience of the ligation didn`t seem to improve!
> Any sugestions?
>
> Thank you
> Torsten
No. It is not necessary to dephosphorylate the target vector prior to
ligation. Lots of people dephosphorylate and lose money on bets to those
who don't. Dephosphorylation does reduce the background re-circularization
of the target vector somewhat, though I have not had any significant
problem with it.
Make sure that the INSERT is somewhere around 3X higher MOLAR concentration
than the TARGET vector for the ligation reaction. I also know that,
depending on your DNA fragment purification technique, TBE buffer inhibits
the ligation reaction and TAE buffer doesn't. Purify your DNA fragments
from TAE gels.
Oh, yeah, and another thing. Molecular biology sucks.
Duke
******************************************************************************
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