Dnase digestion and PCR - (Apr/01/2009 )

Hi,

I had designed primers in the coding region of my gene and checked for the expression by RT-PCR. I just realised a few days back that I forgot to treat the RNA with DNAse.

I got a very fant band when I did PCR using cDNA whose RNA was not treated with DNase. However, after treatment with Dnase, I get a much stronger band?

-SF_HK-

I had designed primers in the coding region of my gene and checked for the expression by RT-PCR. I just realised a few days back that I forgot to treat the RNA with DNAse.

If the primers are on the same exon you can't tell the difference between the gDNA and the cDNA. Always design them on different ones to minimize gDNA contamination.

I got a very fant band when I did PCR using cDNA whose RNA was not treated with DNase. However, after treatment with Dnase, I get a much stronger band?

Usually it is the opposite. If you have gDNA contamination you will have more template.

-molgen-

Do you think its absolutely necessary to treat my RNA each time to Dnase ? I use Trizol for RNA extraction.

-SF_HK-

I did pcr amplification, I usually do the negative control, never i got any bands on negative control.
This time i am getting a faint band around 1kb.

I've changed the water, DNTPs, primer stock, even the polymerase, tubes, tips.

I am seeing the weird situation,

atleast I ran the control 7 or 8 times in duplicate with 2 different sets of solution,
One time I am getting the band and the other time no band
this happens only in negative control. Some of my pcr proudcts size at 700bp, some around 1.5kb they get amplified fine . I think my products are ok. I am really concerned about the negative control since 3 of my pcr product around 1kb.
I think it must be the pcr machine
Thanks in advance
Help me out

-annaramiah-

annaramiah on Jun 22 2009, 02:32 PM said:

I think it must be the pcr machine

Well, your cycler can not produce DNA, when there is none. Most of the time contaminations are PCR products that might even contaminate as aerosols.
It's of much advantage if you are able to seperate pre-PCR and PCR spatially. We have a extra pre-PCR room, where we store our stock solutions (DNTPs, Taq etc) and mix our PCR. The PCR template is then added in the PCR room.
Generally DNA is not allowed to enter the pre-PCR room (apart from our own...) and the room is put under UV-light once a week. Since then we have never had a contamination problem again.
good luck!

-littleaxt-