Q] Digestion,Elution,Ligation

Jun Zhang junz at sfu.ca
Fri Jun 19 12:32:34 EST 1998


Hi, there,

why not try the easy and simple method: double digest->run LMP gel->cut
the bands->melt(70C)->freeze(liquid N2)->thaw(room
t)->spin(5min)->ligation.

isn't that fun.

mark

> > 
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> 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
> 
> 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?
>     In addition, I  heard that the eluted DNA is hard to be ligated by
> DNA ligase.
>     Do you have any idea about these?
> 
> --
> 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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> <HTML>
> <FONT SIZE=-1>Hi~</FONT>
> <BR><FONT SIZE=-1>I'm doing</FONT><FONT SIZE=-1></FONT>
> 
> <P><FONT SIZE=-1>&nbsp;&nbsp; 1.Enzyme digestion, 2 Elution of&nbsp; digested
> DNA fragment , 3. Dephosphorylation,and 4. Ligation</FONT><FONT SIZE=-1></FONT>
> 
> <P><FONT SIZE=-1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; I did the whole step in&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<B>
> 1->2->3->4.</B>.order.</FONT>
> <BR><FONT SIZE=-1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; However
> what if I do&nbsp;&nbsp; in&nbsp; <B>1->3->2->4</B>.</FONT>
> 
> <P>Question1.>>
> <BR><FONT SIZE=-1>&nbsp;&nbsp; What is more efficient ?</FONT>
> <BR><FONT SIZE=-1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<B> #1.Dephosphorylatiion
> before Elution&nbsp; or #2. Dephosphorylation after Elution</B></FONT><FONT SIZE=-1></FONT>
> 
> <P>Question2.>><FONT SIZE=-1></FONT>
> 
> <P><FONT SIZE=-1>&nbsp;&nbsp;&nbsp; I did Elution by using BioRad Elution
> KIT. and I found that about 1/10(or less than 1/10) DNA was eluted.</FONT>
> <BR><FONT SIZE=-1>&nbsp;&nbsp;&nbsp; The efficiency is too low. Is this
> low efficiency is in general?</FONT>
> <BR><FONT SIZE=-1>&nbsp;&nbsp;<B>&nbsp; In addition, I&nbsp; heard that
> the eluted DNA is hard to be ligated by DNA ligase.</B></FONT>
> <BR><B><FONT SIZE=-1>&nbsp;&nbsp;&nbsp; Do you have any idea about these?</FONT></B><FONT SIZE=-1></FONT>
> 
> <P><FONT SIZE=-1>--</FONT>
> <BR><FONT SIZE=-1>Hyunmin Kim</FONT>
> <BR><FONT SIZE=-1>Laboratory of Microbiology</FONT>
> <BR><FONT SIZE=-1>Department of Biology</FONT>
> <BR><FONT SIZE=-1>College of Natural Science</FONT>
> <BR><FONT SIZE=-1>Hanyang Univ.</FONT>
> <BR><FONT SIZE=-1>Haengdang-dong 17&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
> Fax&nbsp;&nbsp; : 082-02-290-0952</FONT>
> <BR><FONT SIZE=-1>Seoul, South Korea&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
> E-mail: vurk at hymail.hanyang.ac.kr</FONT>
> <BR><FONT SIZE=-1></FONT>&nbsp;</HTML>
> 
> --------------55CB9A967621D361223C6674--
> 
> 
> 




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