Ultrasonic Ligation

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Aug 11 13:25:57 EST 1993


arosenth at crc.ac.uk (Dr. A. Rosenthal) writes:

> ... Duncan at genesys.demon.co.uk ("Dr. Duncan Clark") writes:

>> I tried the NAR method using a small Ultrasonic cleaning bath (Jencons
>> Litd., UK, 80W, 40kHz). I tried ligating 1ug EcoR V cut lambda for blunt and
>> 1ug Hind III lambda for sticky ended ligation. In a 15 minute sonication at 16C
>> no ligation was apparent with the blunt and only slight with the sticky ie
>> no more than non-sonicated controls. I was not impressed and haven't tried
>> it again. Maybe my cleaning bath was not the right sort. 
>> 
>> Duncan Clark
 
: Duncan, although it may seem obvious, may I ask whether you tried to transform
: the same ligation reaction after standard o/n ligation at 16`C for comparison.
: IMHO this would be the ultimate proof for failure or success.

This may not be the correct "ultimate" test since the lambda fragments will now
be scrambled and transfection of E. coli with the phage DNA will be a poor
indication of ligation efficiency. I assumed from the above experiment that the
DNA was examined on an agarose gel for increase in DNA size. Well... I also think
that like many other quick procedures, the ultra-fast ligation will save you 5
or 10 minutes today and then cost you several days of work later while you try
to figure out what went wrong with the fast technique ;-)

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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