Amplification of gene cassette

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AXian

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Jan 14, 2010, 4:17:09 AM1/14/10
to Mobile Genetic Elements
I am a biotech student who r just involve in integrons study. I m
currently facing problem amplifying the gene cassette after screening
the presence of integrase gene. I keep getting multiple bands when
using primers that anneal the 5' and 3' conserve region. The PCR
condition between samples are hard to be optimized. Can anyone give me
some advice regarding the problem i m facing?? What condition should i
use? Or should I change the method for detection of gene cassette?

Thanks.

Doval Edouard

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Jan 14, 2010, 8:12:06 AM1/14/10
to mobile-gene...@googlegroups.com
You may have several integrons of a same class in each one of your strains (especially if these strains, related, share a similar plasmid carrying both of these integrons).
Try to sequence the main PCR products to check your primers, in your conditions, targetted the integrons. Do not forget that the "3'CS" is not really well conserved. Maybe try to reduce the time of elongation, to select in a first step, only the smallest integrons. 
Good luck
ED

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Alexandra Moura

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Jan 14, 2010, 7:21:06 PM1/14/10
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If optimization of PCR conditions goes with unsuccessful improvements, the
best way (but not the cheapest...) is to clone the PCR fragments generated,
so you can obtain one fragment per clone.

Even so, depending on the fragment length and amount of secondary
structures, cloning of gene cassettes may also be frustrating.
Take that into account to choose an appropriate vector.
Although I never tested them, my guess is that linear vectors would be more
appropriate for this type of structures.
But, if resources are a limitation, I would try first with a common vector.

A cheaper alternative to cloning is to extract the bands directly from the
gel and purify them to remove the agarose. But this procedure has usually
lower efficiency in obtaining sufficient amount of DNA for sequencing.

So, as you, I'm also looking for the best way to do it.
It would be nice to hear some more tips :)

Anyway, hope this helps!

Best regards,

alexa

mark Toleman

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Jan 20, 2010, 8:01:20 AM1/20/10
to Mobile Genetic Elements
Dear Axian
multiple integrons in individual isolates is a common occurance in
Gram negatives. It is likely that your multiple bands are valid and
represent different variable regions associated with the integrons. We
routinely sequence bands directly from the gel and get good sequence
info. I recommend that you cut each band out of the gel individually-
gel extract using a qiagen kit or similar and then just send to
sequence with conserved sequence primers.
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