A number of possible problems come to mind:
1) It seems most likely that there are inhibitors in your cell pellets. It
also occurred to me that it is possible that your bacteria are not
breaking open (gram positives are often not lysed by standard lysis
solutions) but as B subtilis is autolytic, just letting the culture sit
for long enough should result in adequate lysis for PCR (after all, you
only need one copy!)
I would actually suggest making a genomic DNA prep (using phenol, etc.)
and using this as template for amplification.
2) Also, do you have a positive control? Sometimes primers just don't work
in PCR for no apparent reason. I would also suggest trying a few other
primer pairs.
3) Perhaps the strain of B. subtilis you are using does not contain
the gene in question. Make sure you are using the strain which was
used to do the sequencing project. You may want to obtain the
strain from the sequencing project to make sure.
4) Another trivial explanation is that the organism you are working with
is a contaminant and not B. subtilis. Is the morphology typical?
Glen Tamura
On Wed, 13 Jan 1999, Mark Payton wrote:
>> Dear All,
>> This is aimed at those molecular biologists working with these bacteria. I
> am having difficulty amplifying a gene identified from the whole genome
> project.
>> Are there any tricks to PCR amplifying from these bacteria? Such as DMSO
> etc.(I had no joy with a 6percent solution).
>> I am trying to amplify from cell pellets. Has anyone had success
> amplifying from bacterial pellets? I heat to 95 degrees C for 5 mins prior
> to the PCR cycle.
>> Does anyone have a cDNA library perhaps from B.subtilis that I could have
> an aliquot of to try to PCR from this?
>> I have contacted the people involved in the whole genome project and they
> do not have the relevant DNA to distribute as a shot gun sequencing
> strategy was employed. Bit of a downfall with the whole genome project
> perhaps.
>> I would be extremly grateful for any help.
>> Much thanks,
>>> Mark
>> ------------------------------------------
>> Dr.M.Payton,
> Department of Pharmacology,
> University of Oxford,
> Oxford.
> OX1 3QT
> U.K.
> Tel; +441865 271595
> fax; +441865 271853
> email; mpayton at worf.molbiol.ox.ac.uk>> ------------------------------------------
>>>>
