qRT-PCR and DNA contamination

Analia Alet via methods%40net.bio.net (by analia_alet from intech.gov.ar)
Thu May 22 15:19:31 EST 2008


Hi Emad, for DNAse treatment I have employed TURBO DNA Free form Ambion (cat # 
1907) and it worked well for me. Perhaps your problem is not the DNase. 
Perhaps you have two many DNA/ ug RNA. This kit, for example, removes up to 50 
ug DNA/ml RNA. If there is mor DNA in the sample, the manufactures advise to 
do a rigorous DNAse treatment, were you use more DNAse, adding it in to steps 
to the reaction mix and you duplicate the reaction time.

Another thing to wonder: are you DNAse working well?
Also you can optimize the digestion times with your owun sample and your own 
kit. Perhaps you need to treat the samples for more time. Remember that kits 
are standars, and sometimes you have to adjust them to your own conditions.

Here it is the protocol we used:

Reaction mix:
20ug RNA
10ul 10X Buffer
2ul  TURBO DNA-free
up to 100 ul  water

incubate 30 min 37ºC
add 10 ul DNAse inactivation reagent
incubate 2 min at room temperature (mix 2-3 times by inversion)
ccentrifugate 15 min 10000g 4ºC
SN to a new tube
Store -80ºC

Hope it works for you
Good luck
Best regards
Analía




Mensaje citado por methods-request from oat.bio.indiana.edu:
Hello,

I am doing real time PCR for analysing my genes expression under different
condition of my fungus. We use SYPR green. Actually I isolated RNA using
both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on
column following the kit's instruction. Unfortunately I could still detect a
DNA contamination!! I treated the RNA again with DNase from NEB but still
some traces of DNA exist. I tried to avoid the DNase treatment because I
learned from some people who deal with RT-PCR to avoid DNase treatment and
used Intron Span primers, and here is the big suffering, I used 18-20mere
primers which locate just in the middle of exon borders and still these
primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and still
get some amplification, isn't that terrible? I thought to go to 3 bases at
the 3' end of the primer but then you don't get any good prime which suit in
this area!!? 

Can somebody please give me some hints or has any experience in such a good
DNase which doesn't leave any DNA traces?! I am not optimistic in that. 

Emad 



More information about the Methods mailing list