Why single colony?
lyoung at student.usyd.edu.au
Tue Mar 18 07:45:46 EST 2003
I think the main reason is that during transformation sometimes more than 1 DNA
are transformed into a single cell. Although later they will eventually be
separated into different clones due to incompatible replication origin, the
recovering incubation time is normally only sufficient for less than one cell
division, which may not be sufficient for complete separation, and result in
hybrid colonies. However, this situation rarely occurs and keeping the original
colony should work fine.
"EdiSon, Fang-Hsin CHANG" wrote:
> Thank you for the reply, Ben. The puzzle is that after identifying and
> creating the master plate of the correct clone, why still bother picking up
> a single colony for each innoculation, instead of just taking a swap of
> cells from that master plate and innoculate? Is there any references that
> can explain this as why it was initially proposed as a standard practise? I
> couldn't find any explaination in "Molecular Cloning". cheers! EdiSon.
> ----Original Message Follows----
> From: "Lewis, Ben (FMC)" <Ben.Lewis at fmc.sa.gov.au>
> To: "'chang_edison at hotmail.com'" <chang_edison at hotmail.com>,
> methods at hgmp.mrc.ac.uk
> Subject: RE: Why single colony?
> Date: Fri, 14 Mar 2003 11:53:35 +1030
> Hi Edison,
> Basically you need to create a master plate of one correct clone for further
> use. The first plate after transformation can sometimes have irregular
> clones. That is, not all colonies contain the desired insert. What can
> happen is that if after incubation you receive a lot of colonies, the
> antibiotic concentration in the plate can drop thus, allowing undesired
> colonies to grow without the necessary vector. This is just a method to
> ensure that every time you create a plasmid prep, you effectively have the
> exact same clone. Hence, validating that data you collect is comparable.
> Benjamin Lewis BSc (Hons) GRACI
> Research Associate
> Department of Clinical Pharmacology,
> Level 6, Flinders Medical Centre,
> Flinders University School of Medicine,
> Bedford Park,
> South Australia 5042,
> T?+61 8 82044795
> F?+61 8 82045114
> E ben.lewis at fmc.sa.gov.au
> W www.flinders.sa.gov.au
> -----Original Message-----
> From: chang_edison at hotmail.com [mailto:chang_edison at hotmail.com]
> Sent: Thursday, March 13, 2003 10:11 PM
> To: methods at hgmp.mrc.ac.uk
> Subject: Why single colony?
> After identifying the positive clones from the cell transformation
> plates, why do we still need to streak and pick single colony for DNA
> miniprep and protein? Don't all the decedents from that single
> positive clone have the same genetic made-up? What's the things we
> try to avoid? Mutation in the cloned gene? Hetergeneous mixture of
> plamids? What? Please be kind and blow up my super small brain. Thank
> you for now! Cheers, EdiSon.
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