How can I get rid of the DNA contamination when prepare my total RNA sampls for - I used the RNAqueous-4PCR (isolation of DNA free RNA kit)kit,still hav (Aug/15/2007 )
Hi, all
I used the RNAqueous-4PCR (isolation of DNA free RNA kit)kit, conducted the exprement as the instruction of the kit.
1.add 0.1 volume of 10X DNase 1 buffer and 1ul of DNase 1(per 100ul RNA solution )
2.incubate 20min at 37C
3.add 0.1 volume DNase inactivation reagent
.....
then I used the RNA samples as the PCR template to run PCR(S15 primer), and there were PCR products,so it meant that there was genome DNA contamination, how can I deal with this DNA contamination problem at the situation,Thank you all!
Jane
did you quick check your sample before DNase ? i mean a spectrum 230-->280 would tell you if contaminants remain. If ethanol remains for ex, DNsase will be less efficient.
other way, ask kindly for a replacement of the DNase (+ buffer) to the comany.
Or bought an other DNase. i love ambion's turbo one...
I used the RNAqueous-4PCR (isolation of DNA free RNA kit)kit, conducted the exprement as the instruction of the kit.
1.add 0.1 volume of 10X DNase 1 buffer and 1ul of DNase 1(per 100ul RNA solution )
2.incubate 20min at 37C
3.add 0.1 volume DNase inactivation reagent
.....
then I used the RNA samples as the PCR template to run PCR(S15 primer), and there were PCR products,so it meant that there was genome DNA contamination, how can I deal with this DNA contamination problem at the situation,Thank you all!
Jane
Depending on the tissue or substance from which you are extracting RNA, you may need to treat the RNA with DNAse twice. I've found this to be the case with intestine tissue.
I always treat 2X with DNase to make sure I get rid of all DNA
Thank you for all of your help!
other way, ask kindly for a replacement of the DNase (+ buffer) to the comany.
Or bought an other DNase. i love ambion's turbo one...
Thank you very much that you let me notice I should take 260/230 ratio into account
my samples' 260/230 are very low,about 1.51~0.1,I don't know why the ratio is so low even if I followed the Ambion's manual so tightly .
should I precipitate the RNA samples and use DNase again?
or
What should I do to optimise the procedure of that kit to avoid re-precipitate the sample,because my samples are very limit, thank you so much for your help!!!Jane
ok well critical step is the drying of the RNA. Typically i carefully spin the ethanol and pipett all i can pipett, and heat 65° 10' on a thermoblock with a cover (to minimize the dust to enter in the tubes). that's for ethanol part of 230.
I do 2 washes with 70% etoh and carefully detach the RNA pellet from the side of the tube.
Helps removing salts, which btw reduces the 230 value.
check few threads on this (here for ex)
I do 2 washes with 70% etoh and carefully detach the RNA pellet from the side of the tube.
Helps removing salts, which btw reduces the 230 value.
check few threads on this (here for ex)
Thanks,I will try.
and did you ever see the protocol of RNA precipitation of the RNAqueous-4PCR kit(Ambion),it uses Ammonium Acetate ,Linear Acrylamide and 100% ethanol mix well -20C overnight then centrifuge the tube ,resuspend the RNA in elution solution . I don't know why it doesn't use 70% ethanol to wash the pellet . And if don't use 70% ethanol ,will the RNA has more ethanol remain?
Thanks. I used the precipitation protocol , then use Dnase treat it again, and still have DNA contamination.
Oh,I go crazy about it !!
I do 2 washes with 70% etoh and carefully detach the RNA pellet from the side of the tube.
Helps removing salts, which btw reduces the 230 value.
check few threads on this (here for ex)
Thanks,I will try.
and did you ever see the protocol of RNA precipitation of the RNAqueous-4PCR kit(Ambion),it uses Ammonium Acetate ,Linear Acrylamide and 100% ethanol mix well -20C overnight then centrifuge the tube ,resuspend the RNA in elution solution . I don't know why it doesn't use 70% ethanol to wash the pellet . And if don't use 70% ethanol ,will the RNA has more ethanol remain?
Thanks. I used the precipitation protocol , then use Dnase treat it again, and still have DNA contamination.
Oh,I go crazy about it !!
You might like to try qiagen off colume DNase treatment and then clean up protocol. I have the same DNA contamination for my RT-PCR now.