From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: cm amplification? (FAQ!!)
Summary: FAQ list construction
Keywords: FAQ list
Date: 30 Sep 92 16:57:58 GMT
Followup-To: Chloramphenicol [FAQ]
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

................................................................................
>Can anyone out there supply me with a protocol for chlorampenicol
>amplification of pUC-based plasmids in E. coli?
................................................................................

Due to the recurrence of the chloramphenicol question,
I've started the construction of a FAQ list for
bionet.methds-reagnts and would like other people to
contribute to it's growth and evolution. Please feel free
to include what you like and any suggestions for a better
FAQ list. Thank You. -Paul.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
voice: (301) 846-5581
FAX: (301) 846-5598
e-mail: pnh@ncifcrf.gov

--------------------------------------------------------------------------------
I think a simple format of questions and answers is a good start since I
haven't seen or heard of a FAQ list at all for this newgroup. Perhaps for
simplicity, I should ask for an ASCII format of not more than 80 characters
in line length. Please include references if possible. What do you think an
appropriate reference format should be? They can be provided as J. Bacteriol.,
Medlars, BiBTeX, etc.. If the list grows too large, it may need to be separated
into catagories such as  1) General information 2) PCR technology
3) Hybridization 4) Plasmids ..etc. 


The FAQ list could be in the form:

.........+.........+.........+.........+.........+.........+.........+.........+
        10        20        30        40        50        60        70        80


This is the FAQ list prototype for bionet.methds-reagnts
Last update was on 29 September 1992


[Note: Please add any relevant or missing information and return to me at
the internet address pnh@ncifcrf.gov]

================================================================================

What are some good reference books for methods in molecular biology?

There are many books which provide details and recipes for molecular
cloning techniques. The following is a list of reference books which
are commonly available...

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.

Miller, J. H. 1972. Experiments in molecular genetics, p.431-435. Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York.

================================================================================

Where can I get information about the genotypes and phenotypes of common
E. coli strains used in molecular genetics?

The following provide detailed descriptions of E. coli strains...

Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia
coli K-12. Bacteriol. Rev. 36:525-557.

Bachmann, B. J. 1990. Linkage map of Escherichia coli K-12, Edition 8.
Microbiol. Rev. 54:130-197.

================================================================================

How can I access MEDLINE?

First, you'll need to get an account on MEDLARS. Call the MEDLARS Management
Section Service Desk: 800-638-8480. Once you fill out the Online Billing
Agreement with the NTIS, you get a userid and password from MEDLARS.

Unless you're quite expert with MESH terms and MEDLINE searching, you should
order a copy of GRATEFUL MED 6.0. You can then formulate your search offline
and then telnet to medlars.nlm.nih.gov

If you haven't got telnet, you can access MEDLINE via BITNIS and get your
MEDLINE searches returned via email. You can do searches in command format or
by using Grateful Med 6.9 and a program called smed.

You can also access Medline by way of Paperchase, a dial-up service available
through a modem. They provide a 24 hr. toll-free number for questions.
In the U.S. you can call 800-722-2075. From Canada, you can call
collect 617-278-3900. You can also access Paperchase through
telnet to biotechnet.com if you open an account with them first. The
number for BioTechNet is 508-655-8282. You can get more information
about this by sending e-mail to biotechnet@biotechnet.com.

================================================================================

How do I amplify pBR322 plasmid using chloramphenicol?

This is the procedure to amplify pUC and pBR based vectors for
large scale plasmid preps. The procedure is directly from Maniatis:

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.

Innoculate a colony from your plate into 10ml of LB broth containing
an appropriate antibiotic, probably ampicillin for pUC plasmids.
Other rich media such as 2xYT, as well as minimal media can also be used. 
Incubate with shaking at 37 degrees overnight.

The next morning add 0.1ml of your overnight culture to 25ml of LB
with  antibiotic (ampicillin) in a 100ml flask. Incubate at 37 degrees
with shaking until the culture is at late log. Usually a couple of
hours  is enough. Add 25ml of your fresh late log culture to 500ml of LB
with antibiotic (ampicillin) which has been prewarmed to 37 degrees.
Use a 2L flask for good aeration. Incubate with shaking for exactly
2.5 hours at 37 degrees. Add chloramphenicol to the culture to a final
concentration of 150ug-170ug/ml. If you use spectinomycin the final
concentration should be 300ug/ml. 30ug/ml of chloramphenicol can also
be used. This seems to work just as good as the higher concentration.
Incubate with vigorous shaking for 12-16 hours. If your timing is OK
an overnight incubation will do. Don't worry if the culture turbidity
drops during this incubation. But don't leave the culture for much
longer or you may get too much cell lysis and plasmid yield will drop.
Spin your culture and isolate the plasmid by conventional methods.

There is a mutation in pUC plasmids that increase copy number, but you
can still amplify the plasmids. Most cloning vectors (pUC and pBR
included) have a relaxed replication and don't use the short lifetime host
proteins used in host chromosome replication. So by adding antibiotics
such as chloramphenicol or spectinomycin (which inhibit protein
synthesis) you stop chromosomal replication and cell division without
stopping plasmid replication. The end result is greatly increased
plasmid yield.

================================================================================