Q] Digestion,Elution,Ligation

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Jun 19 12:21:59 EST 1998


Hyunmin Kim wrote:
> 
> Hi~
> I'm doing
> 
>    1.Enzyme digestion, 2 Elution of  digested DNA fragment , 3.
> Dephosphorylation,and 4. Ligation
> 
>       I did the whole step in          1->2->3->4..order.
>          However what if I do   in  1->3->2->4.
> 
> Question1.>>
>    What is more efficient ?
>       #1.Dephosphorylatiion before Elution  or #2. Dephosphorylation
> after Elution

No difference, if you make sure your phosphatase is completely inactivated
before addition of the other DNA and ligase. You can use shrimp alkaline
phosphatase that is easily inactivated by 65C incubation, but many people have
found that some batches of SAP are lousy. If you use CIAP, you´ll have to
phenol/chloroform-extract the DNA at least twice to get rid of the
phosphatase. In that case, it might also be better to do the dephosph before
the gel run (to remove additional phosphatase activity)


> 
> Question2.>>
> 
>     I did Elution by using BioRad Elution KIT. and I found that about
> 1/10(or less than 1/10) DNA was eluted.
>     The efficiency is too low. Is this low efficiency is in general?

10% recovery is unacceptably low. Most techniques give 50%+x recovery, agarase
up to 98%. Try a different elution method (e.g. agarase digestion of low
melting point agarose gel slices). Call several companies for test kits. They
give them for free, and you can try them out to find the one that fits your
needs best.


>     In addition, I  heard that the eluted DNA is hard to be ligated by
> DNA ligase.
>     Do you have any idea about these?

Yes, some elution methods (especially the most well-known) tend to ruin your
DNA. Although you have a nice band on a control gel, your DNA will NEVER be
cloned. Sometimes contaminants from the agarose are present in the final DNA
and will inhibit ligation. Sometimes the problem will never become clear. As
above, try several methods to find the best for you. 

Best regards,
Frank



> 
> --
> Hyunmin Kim
> Laboratory of Microbiology
> Department of Biology
> College of Natural Science
> Hanyang Univ.
> Haengdang-dong 17                Fax   : 082-02-290-0952
> Seoul, South Korea               E-mail: vurk at hymail.hanyang.ac.kr
>



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