strange cloning result

Hiranya S. Roychowdhury hroychow at nmsu.edu
Tue Mar 28 12:15:21 EST 2000


Usually, the hi background is due to self ligation of the vector if it was
not dephosphorylated. If u checked the plasmids on the control plate, u'll
find that most have uncut plasmids showing larger size. Although there is
the popular practice of not bothering to dephosphorylate vectors w/
non-identical sites, concatenation is very real in such cases (intra-mol.
ligation is always favored over inter-mol.)
The other possibility is that one of the enzymes did not cut 100% of the
vector molecules. This is rare, given sufficient time for digestion
(especially w/ those two).
As for the low number of transformants in v+i plates, it is hard to
speculate. However, high i:v ratio has nothing to do with it. In fact, in
your case, it probably helped you get some positives by forcing the v into
accepting some i's. What does happen, in certain situations, as you rightly
mentioned,is that high molar conc. of the i results in i concatenation. I
think that IF the p-i is toxic to the cell in native form, the multiple i
allows the host to maintain the plasmid. 
To check if you do have multiple I, digest w/ one RE and check the size of
the linearized plasmid.

At 10:51 PM 3/27/00 +0100, Wolfgang Schechinger wrote:
>Hi all, 
>
>recently, I performed a simple cloning experiment (sticky 
>HindIII-XbaI insert into pCDNA3). 
>As directed in the textbooks, I had an control ligation without the 
>insert. This control gave me about 300 colonies as "background". The 
>sample containing the insert yielded 100 colonies only of which I 
>prepped 24. Finally 15 (60%) contained the insert though I didn't 
>expect to get any positive. Is there any reasonable explanation for 
>this except statistics? Was it an insert overdose killing most bugs 
>due to too long insert concatenations? (Concentrations were estimated 
>on gel, insert to host ratio was 5)
>
>Wolfgang
>-----
>This message is encrypted. Use your brain to decode it.
>-----
>Dr. Wolfgang Schechinger, Dept. of Pathobiochemistry
>University of Tuebingen, Germany
>email: wolfgang.schechinger at med.uni-tuebingen.de 
>wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
>-----
>usual disclaimers apply 
>-----
>---
>


Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu

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