Ligations-HELP

Sailesh Surapureddi SaiSu at MCB.LiU.SE
Thu Jul 6 11:02:47 EST 1995


>To: methods-and-reagents at net.bio.net
>From: tkuwada at neuron.uchc.edu (TimHusky)
>Subject: Ligations-HELP
>Date: 6 Jul 1995 02:52:28 GMT
>
>Iam having trouble with a seemingly straight forward sticky-end
>ligation.  I have 
>used up to 800U/20ul rxn with Insert:plasmid ratio's of 3:1  - 13:1,
>total DNA of 
>up to 300ng. These rxn have been overnight @ 16C.I have several
>questions:
>
>1) Am I using too much or too little DNA?
:		No, you are not using too much, but why waste insert and plasmid, reduce it 
two 2:1 ratio, besure to denature them both before adding the ligase buffer and 
enzyme.

>2) Should I clean my DNA from LMP gels?  I have tried using DNA in LMP
>and cleaning it w/ a promega kit (bad yield)

:Yes, generally I clean my DNA with Qiaex II ( gives  good yeild)

>3) I am using 400U/ul T4 ligase (NE Biolabs), has anyone else had any
>problems w/ their ligase or buffer?

:No, no problems so far.

>4) Will spiking my buffer w/extra ATP help & what is the deal w/ PEG,
>does it increase ligation efficiency

:No, again, it does not increase the ligation efficiency. But you can clean the 
ligation mix with Et-OH for added safety for transformations.

>5) How can I increase the competency of my DH5-alpha cells,Iam
>currently using a 50mM CaCl2 2X wash of cells at .3-.4OD @ 590nm

:The ligations conditions have not been described by you, you could try this 
(incubating the competent cells with insert and vector on ice for 30min and heat 
shock at 42 for 5 min and again on ice for 5 min). That should do the trick.  
>I would greatly appreciate any tips.

One more: the ligation could increased and the time decreased by sonicating the 
ligation mixture in sonication water bath for 15min at 16 C, this works very 
well in my case and now I get lot of colonies.

Hope this helps
Sailesh.
>Tim Kuwada
>Med Student
>Univ Conn Health Center
>tkuwada at neuron.uchc.edu

Contacts:

Dr.Sailesh Surapureddi										voice of lab:+46-13-223917
Post Doc																								voice of Res:+46-13-138839
Institiute of Cell Biology						Fax									:+46-13-224149
Halsouniversitetet
Linkoping, S 581 85
Sweden.



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