TimHusky tkuwada at
Wed Jul 5 21:52:28 EST 1995

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

1) Am I using too much or too little DNA?
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)
3) I am using 400U/ul T4 ligase (NE Biolabs), has anyone else had any
problems w/ their ligase or buffer?
4) Will spiking my buffer w/extra ATP help & what is the deal w/ PEG,
does it increase ligation efficiency
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

I would greatly appreciate any tips.

Tim Kuwada
Med Student
Univ Conn Health Center
tkuwada at

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