hroychow at NMSU.EDU
Mon May 29 10:47:53 EST 1995
On Fri, 26 May 1995, Aron B. Jaffe wrote:
> All I said was it was "not necessary". The efficiency of sticky end ligations
> should be high enough where one need not worry about CIPing - and the problems
> that may arise from this procedure...
Thank you for the letter. I understand what you are saying. But
that , precisely, is my point: Sticky end ligations are very very
efficient and highly favored. The dynamics of the ligation procedure
dictates that the two closest compatible ends would anneal
fastest/earliest. Thus, intramolecular ligation is favored at a rate
significantly higher than that of intermolecular ligation. In order to
drive the process towards the later, the insert:vector ratio would have to
be very high (7:1 or 10:1), unless the intramolecular event is discouraged
(lack of PO4 end). These days we are finding ourselves working with
amounts of inserts that would have been considered ridiculous even 15 yrs
back. Hence, we try to optimize the conditions where as little insert may
be utilized as is physically possible.
The problem with vectors following CIPing is nothing new. It is
not unique to a few labs, nor does it reflect badly on the researcher's
experimental acumen. I am sure everybody has been faced with similar
situation some time or the other. I know I was, and it was very
frustrating at that time. Then I started being really fastidious about my
vector preps and never had any problem ever since. I am so much sold on
the idea of dephosphorylating the vector that I carry out CIPing
immediately following RE digestion, by just adding the appropriate
zinc-buffer (10x) to the RE buffer. After about an 30 min I clean the DNA
thoroughly as I mentioned. I have been able to clone up to 16kb in pUC19
without much hassle.
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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