Help about Genereleaser

Michelle.Gleeson at SMTPGWY.AGRIC.NSW.GOV.AU Michelle.Gleeson at SMTPGWY.AGRIC.NSW.GOV.AU
Sun Jan 28 18:12:36 EST 1996


     Hi all,
        I have tried to use Genereleaser on total nucleic acid extracts to 
     help in amplifying viroid RNA with reverse transcriptase PCR.  I 
     wasn't using it to actually extract from leaf tissue, but was using it 
     to bind inhibitors present in the dirty extracts.  It did work, in 
     that I could only amplify something from samples which had the GR, but 
     I developed problems with negative controls coming up positive.
         I looked at the stuff under the microscope to try and work out 
     what it was made of, (glassmilk/clay???) and was very surprised to 
     find bacteria swimming around in the matrix.  I plated out 50ul onto 
     Sucrose Peptone agar and LB agar and incubated at 25C for 3 days.  
     There was significant growth, which was identified through fatty acid 
     analysis to be a Rhodococcus, probably R. globerulus.  I made an 
     extract of the bacterial DNA and did standard PCR on it using my 
     primers , and got the same sized band as the negative control bands.   
       The Australian distributor contacted the manufacturer, which said 
     they couldn't isolate anything.  So, we bought another batch.  We 
     opened it in a laminar flow an plated out again.  Same growth, same 
     ID, same PCR products in the negative control.  I don't use it 
     anymore, and haven't for about 18 months now, so they may have 
     rectified the problem.  It is interesting that they don't recommend 
     the product for use in RAPD PCR.  I guess I was unlucky that my viroid 
     primers matched the contaminant.
        Other people in my lab use GR happily, so it couldn't hurt to try 
     it.  Ask for a sample first and plate some out to check, just to be 
     safe.




More information about the Virology mailing list