hybridisation

chhavi porwal via methods%40net.bio.net (by chhavi.porwal from gmail.com)
Wed Mar 23 10:33:01 EST 2011


Dear All,
Myself a PhD student in department of Microbiology All India Institute of
Medical Sciences New Delhi.my query is about standerdisation of RLBA?
In my lab, we are doing RLBA for RIF (rpoB gene) of M.tuberculosis last 5
years, (paper, suresh et al). now i am starting to doing RLBA for gyrase of
M.tuberculosis, but i am facing some problem, enlisted below.
1) i am getting mutatn type signals in H37Rv strain?
2) i am getting good results in resistant strains (confirmed by
sequencing).as i am getting loss as well as gain of specific mutation.
3) i am getting the same signals in sensitive strains also (as i am getting
in H37Rv)

WHAT COULD BE THE REGION?

i have done many changes during standardization.
At present my working conditions for RLBA are

1) PCR annealing tem. is 55 C
2) RLBA incubation temperaturs is 80 C ( i also did it with at 60 C, but the
results are same)
3) i have used hot start taq DNA polymerase also, but the results were same
4) i have choose published primer which amplify the hot spot region of
M.tuberculosis


Kindly suggest me, what should i do now?
as i am getting good results in resistant strains but in sensitive and
stranded strains, why the mutant type signals are present? what could be the
reason?
Kindly GUIDE me......

Warm Regards Chhavi


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