Looking for plasmids

[MJo]

Dear all,

I have a group of 169 isolates and I wanted to screen the presence of
plasmids among this collection. I was wondering if any of you knows an
accurate and fast method to do such screening, without the need of
DNAp extraction.

Thank you for your attention.

Best regards.

MJo

[Doval Edouard]

Dear MJo, I think you should try the PCR colony ; if you need  a protocol, feel free to ask me. in a plate you can test 96 isolates. good luck Integronboy --- En date de : Lun 13.10.08, MJo <maria...@gmail.com> a écrit :

[Christopher Thomas]

Dear MJo
By definition plasmids are extrachromosomal. You can find plasmid DNA
integrated into the chromosome but these are not plasmids. So if you
want to show that there are plasmids you need to isolate plasmid DNA, or
at least separate it from chromosomal DNA as in the Eckhardt technique.
If you simply want to test for the presence of sequences related to a
particular plasmid group then you can use PCR but that will not prove
that you have a plasmid of that sort there.
Best wishes
Chris Thomas

Christopher M Thomas
Director of the University Graduate School
Professor of Molecular Genetics
School of Biosciences
University of Birmingham
Edgbaston
Birmingham
B15 2TT

Phone +44 121 414 5903
Fax +44 121 414 5925

[Maria Carvalho]

Dear Dr. Thomas,

 

Thank you for your suggestions and explanations.

 

I want to screen my isolates for the presence of plasmids, to see whether or not an isolate possesses plasmids. I was wondering if there is any technic (besides observating gel electrophoresis of total DNA) that enables me to do such screening without doing extraction of the plasmids.

It looks like I have to do the extraction.

Once again, than you for your help.

Kind regards.

MJo

2008/10/14 Christopher Thomas <C.M.T...@bham.ac.uk>

--
Maria João Mendes de Carvalho
Molecular Biology Lab
CESAM | Biology Department
Campus Universitário de Santiago
University of Aveiro
3810-193 Aveiro | Portugal
mjcar...@ua.pt
maria...@gmail.com
http://sweet.ua.pt/~f426
Telf: +351234370970
Fax: +351234372587

[Maria Carvalho]

Dear Integronboy,

 

I think that I didn't understand your suggestion... At the beginning I want to look for plasmids in general and not assign them to a specific group. I think that with the PCR approach I will be directing my research to specific groups, as I will have to use primers for a specific fragment. Isn't that so?

 

Thank you for you help.

Kind regards.

MJo

 

2008/10/14 Doval Edouard <dovale...@yahoo.fr>

[Christopher Thomas]

Dear MJo

The Eckhardt technique allows you to lyse the cells in the gel well and then separate chromosomal DNA from plasmid DNA without doing the isolation, but as you say you still need to do the electrophoresis – but it has the advantage that it does not depend on the plasmid DNA being ccc.  This would be a good way to do an initial screen.

Best wishes

Chris

 

Christopher M Thomas

Director of the University Graduate School

Professor of Molecular Genetics

School of Biosciences

University of Birmingham

Edgbaston

Birmingham

B15 2TT

 

Phone +44 121 414 5903

Fax +44 121 414 5925

[Maria Carvalho]

Dear Chris,

 

Thank you very much for your help.

 

I am already looking for the protocol on the internet. 

Maybe, this will be my choice!!

 

Thank you, again.

 

Best regards.

[Christopher Thomas]

The original reference is Plasmid 1, 584-588 – that was 1978.

[mark]

Dear MJo,
Just joined the group so probably a little late--I have recently got a
method working to do just this which involves a PFGE technique--the
strains are lysed in gel like a normal PFGE typing experiment and the
treated with S1-this nicks single stranded DNA within CCC and plasmids
then run true to size (ref Barton et al 1995 Anal Biochem 226
235-240). I have found that with this method plasmid bands which are
unseen normally become visable with normal ethidium staining. You can
thn perform further characterisation directly on the gel--The gel can
b dried-re-hydrated and then probed which works nicely or the bands
can be cut out b4 or after hybridisation and used as template in a
random PCR experiment to sequence the whole plasmid or just for typing
PCR experiments like Alessandra Carotoli has recently published. Let
me know if you need more detailed protocol.
best wishes.
Mark

[Maria Carvalho]

Hello, Mark!

 

Thank you very much for your suggestion.

I will study the possibilities presented by you and Chris and then, according to my aims I'll decide which is the best one.

Be sure that I'll ask you for more details!!

 

Thank You!!

 

Best regards.

MJo