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SYTO9 staining - Flurometer false positive readings

SYTO9 Flurometer PCR RT-PCR bacteria

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#1 asosman87

asosman87

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Posted 17 June 2012 - 12:01 PM

HI! I recently joined this forum yet did not actively involve due to my lack of English writing skills. However, I decide to step up and hopefully get some opinions from great people.

I am testing newly arrived PCR machine by repeating two step RT-PCR previously done successfully.

In this lab, they are testing the success of PCR by STYO9 staining followed by flurometer instead gel electrophoresis.

If there hasn't been specific amplication of nucleic acids happened, the fluroscence measure should be less than 10, and if there has, the measure is above 10.

My gene of interest was in ribosomal RNA in bacteria, yet there is exact same sequence existing in complete DNA genome of bacteria as well. To see if RT actually happens before PCR, I separated my experiments into two group

1 - PCR followed by RT using total RNA
2 - PCR using total RNA directly (which contain some genomic DNA due to purification problem)

within each group I have four subjects like following

a) primer combination 1
B) primer combination 2
c) positive control (beta actin primers)
d) negative control (exactly same as a) but using water instead RNA)

And... after PCR the flurometer shows this figures

1a) 13
1b) 8
1c) 14
1d) 10
2a) 32
2b) 30
2c) 35
2d) 20

I haven't really detailed my experiments (running conditions) and I can clearly tell from this figure that my RT fails and something during RT disrupt normal PCR process. Yet, my specific concern is

Why negative control in second group shows relatively high meausre of fluroscence? the only difference in first group and second group is RT ( invitrogen superscript 3, 65 degree for 5 min, 55 degree for 30 min and 70 degree for 15min). It could be just my pipetting error or contaminations yet I am curious if there are other people having same false-positive readings in fluroscence mesaure and how they control it.

Thanks!





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