Cloning efficiency vs. vector size.

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Mar 1 21:10:26 EST 2001


Although I have not cloned 17kb, I have cloned 12kb into pGEM3zf and
pGEM7zf. Both are about 3kb vectors.
I seriously doubt that vector size is of any consequence in the ultimate
cloning + transformation. What matters is the total size of a plasmid in
transfomation. The efficiency is almost always inversely proportional to
the final size of the plasmid. Where sizes more than 10kb are involved,
best results are obtained via electroporation. However, up to 20kb may be
successfully taken up by freshly prepared E coli by Hanahan's protocol,
albeit at a low efficiency.



At 08:31 AM 3/1/01 GMT, Tomas Aparicio wrote:
>Good morning/evening:
>
>    I have a basic doubt about cloning eficiency depending upon vector size:
>
>    Using the same replication origin (for instance, a ColE1 derived
>vector), is important the vector size in order to clone very big inserts?
>    And another question: have anybody succeeded in cloning PCR fragments
>bigger than 17 Kb? Which vector did you use?
>
>Thanks in advance, and good luck.
>
>Tomas Aparicio.
>
>
>
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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