-RT contamination problem in PCR - (Apr/19/2016 )
After finishing cDNA synthesis, I immediately ran PCR with -RT and +RT tubes.
There's no problem in +RT band but few bands in cDNA size appear in -RT samples.
No gDNA contamination is detected but I'm now struggling with -RT contamination showing cDNA size band in agarose gel.
The procedure is like this.
First, I set up PCR machine and label tubes and make master mix in RNase/DNase free water -> dNTP mix -> forward and reverse primer -> PCR buffer -> Taq polymerase loading order.
( I load Taq pol right after loading +RT/-RT samples)
Then I load samples in each tube and then load master mix.
I load -RT first and close the lids to prevent the contamination from +RT.
And considering that there is no band in NTC it should not be water contamination.
What should be the matter??
I'm really exhausted .. and my professor is starting to get frustrated about this contamination issue..
I synthesized cDNA 2 weeks ago but still struggling at the quality check step.
A few questions in order to be able to help you.
1. Did you use a designated area to work?, Do you clean thoroughly all materials?, Are you using only dna/dnase/ rnase free materials and reagents?
2. What method you use to extract RNA?
3. Did you did a DNase digestion step?
4. did you ran the RNA in a native or denaturant gel or none at all?
4.Do you add the Taq to the master mix? or to each tube individually?
5. do you open and close tubes for each individual addition? or after you add all?
6. if possible upload a photo of the gel
7. until you answer all these, I would advise to add the MM (with all the reagents) first and then add the samples always beginning with the RT-
merlav on Wed Apr 20 19:49:00 2016 said:
A few questions in order to be able to help you.
1. Did you use a designated area to work?, Do you clean thoroughly all materials?, Are you using only dna/dnase/ rnase free materials and reagents?
2. What method you use to extract RNA?
3. Did you did a DNase digestion step?
4. did you ran the RNA in a native or denaturant gel or none at all?
4.Do you add the Taq to the master mix? or to each tube individually?
5. do you open and close tubes for each individual addition? or after you add all?
6. if possible upload a photo of the gel
7. until you answer all these, I would advise to add the MM (with all the reagents) first and then add the samples always beginning with the RT-
1. Did you use a designated area to work?, Do you clean thoroughly all materials?, Are you using only dna/dnase/ rnase free materials and reagents?
Yes we have pre-PCR dack and clean the pipettes with 1% SDS -> 70% EtOH -> ddH2O and we use only DNase/RNase free water when making master mix.
2. What method you use to extract RNA? We lysis cell pellet with trizol and use Choloroform, Isopropanol, 75% EtOH step to isolate RNA. Then we dissolve the RNA with TE buffer.
3. Did you did a DNase digestion step? Yes our lab always treat DNase1 at the very first step of cDNA synthesis.
4. did you ran the RNA in a native or denaturant gel or none at all?
Before making cDNA I always check RNA quality with 1.5% agarose gel unless the isolated RNA has low concentration. After that, I get confirmation from my professor if the quality is OK
5.Do you add the Taq to the master mix? or to each tube individually? I add Taq to the master mix since our pippette's smallest unit is 0.2~2ul and the Taq amount needed to be loaded to each sample is 0.05ul.
6. do you open and close tubes for each individual addition? or after you add all? I close them after I complete the master mix addition since I use micro tubes which have 8 tubes in a row.
7. if possible upload a photo of the gel
I cannot upload the photo because the site is refusing the URL..
Thank you for your reply!
So, you're doing RT-PCR, but looking at the product on a gel as a quality step and you see a product of the appropriate size in your -RT control. Do your primers span large introns? It's quite possible your DNAse digestion is incomplete. I personally like the ValidPrime method, which lets you assess the degree of gDNA contamination and correct for it. Could be interesting for you: http://www.tataa.com/products-page/qpcr/validprime/
At the gel you should see if there is DNA contamination (a very high weight band) or not, if no dna then you are introducing the contamination at downstream steps. If there is DNA contamination re-digest with dnase the samples. Clean the area with a commercial detergent specifically for RNAase/DNA contamination, what you are using is not enough to get rid of any DNA contamination. If possible have pipettes and tips (with filters) dedicated to RNA only. Add the Taq to the mix, multiple pipetting introduces contamination. When doing the MM add 1st water, then taq, then the primers. Add first the MM to every tube and don't close it until you add the sample. opening and closing multiple times the tubes could make you carry a contamination to other tubes. Change tips often, the cost of repeating an assay is a lot more than the few cents that cost the tips.
It is possible your Dnase step is not enough. Try to add more Dnase enzyme or reduce you template?