Ligation failed (continued)

newsnet customer customer at newsnet.com
Mon May 29 10:29:27 EST 2006


"Jose de las Heras" <josenet at tiscali.co.uk> wrote in message
news:4du1mkF1bcdnaU1 at individual.net...
>
> "newsnet customer" <customer at newsnet.com> wrote in message
> news:pVWdg.11886$S7.3275 at news-server.bigpond.net.au...
> >> > Hi,
> >> >
> >> > I have transformed an insert/gene (2kb) into a cloning vector.
> >> > Then cut the gene out to ensure it has sticky ends.
> >> > Also, I have linearised an expression vector (11kb) with compatible
> >> > sticky ends to the insert.
> >> >
> >> > Here is the ligation reaction:
> >> >
> >> > insert (12ng/ul)                     2ul
> >> > linearised vector (6ng/ul)        4ul
> >> > 10X lig. buffer                       1ul
> >> > T4 ligase                              1ul
> >> > Water                                  2ul
> >> > TOTAL                                 10ul
> >> >
> >> > overnight incubation @ 4C in accordance to Roche T4 ligase
information
> >> > sheet.
> >> > Total DNA in ligation reaction is 48ng.
> >> > insert:vector ratio is 1:1 in accordance to Roche T4 ligase
information
> >> > sheet when insert to vector size are significantly different.
> >>
> >> Actually, no, the insert to vector ratio is ~1:5...unless you are
> >> cutting asymmetrically, then you don't have enough. You have to go by
> >> molar ratio (or the equivalent of dividing mass by size), not mass
ratio.
> >
> >
> > That ratio doesn't look right to me. And please define molar ratio for
> > DNA?
>
> molar ratio is the same whether you talk of DNA or whatever. It refers to
> number of molecules... you must know what a mol of a substance is, right?
>
> the ratio is correct. 1:5.5 to be exact.
>
> Your vector is 5.5x bigger than the insert, therefore if you have the same
> *mass* (24ng in your example) you have 5.5x more molecules of insert than
of
> vector.
>
> If vector and insert ar of the same size, then for a 1:1 ratio you use the
> same mass. If the vector is say, 3x bigger, then for a 1:1 ratio (in
number
> of
> molecules, which is what matters here) you have to use a third of the
amount
> of vector. Think about it.
>
> ---
>
> This never made much sense to me.
> I think he meant that if the vector is 3x bigger then you would use 3x
more
> vector (OR 1/3 of insert) than insert in order to get the 1:1 molar ratio.
>
> Cheers
> Sharp Tool
>
>
> You're right of course... should've proof-read what I wrote. But it shows
> you got it, so... great! :-)
>
> Jose

Thanks for your help :)

Cheers
Sharp Tool





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