correct bands in RT-PCR negative control - (Apr/14/2004 )
hi alex
I had such a problem . when you working on a gene with processed pseudogenes in the genome , you will get bands with same size in no-RT controls . treat the RNA with Dnase! first . after treatment use half of the RNA as a no-RT control and the other half as a usuall RNA for RT-PCR . then compare the bands in 2 samples .
Good Luck
-yaser-
Please forward to the link
http://www.narna.ncl.ac.uk/RT_primers.html
a good message by Professor Kenneth M. Baldwin
-n0248-
QUOTE (alex_osu3 @ Apr 14 2004, 08:06 AM)
Dear all,
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
Hi Alex,
I'm having sort of the same problem as you currently. In my case however, my actin primers (positive control) are giving me the correct sized band in +RT and -RT reactions. I'll post again if I'm close to solving it. Likewise, I'd appreciate any help as well if you've got some suggestions. Perhaps we can help each other solve it this way.
-calavera1984-
QUOTE (calavera1984 @ Apr 21 2008, 12:50 AM)
QUOTE (alex_osu3 @ Apr 14 2004, 08:06 AM)
Dear all,
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
Hi Alex,
I'm having sort of the same problem as you currently. In my case however, my actin primers (positive control) are giving me the correct sized band in +RT and -RT reactions. I'll post again if I'm close to solving it. Likewise, I'd appreciate any help as well if you've got some suggestions. Perhaps we can help each other solve it this way.
This means you have contamination in your -RT reactions. There is DNA in one of your solutions, or is being introduced to your negative reactions through a contaminated pipettor, etc. I would start by cleaning everything, changing your water, and making new primer stocks. (Reorder your primers if you have to). If you have actin bands in your -RT, but no bands for your transcript of interest (assuming you got the transcript band in your +RT), then it's possible that your actin primers have been contaminated.
-MolBioGirl-
QUOTE (MolBioGirl @ Apr 21 2008, 06:38 AM)
QUOTE (calavera1984 @ Apr 21 2008, 12:50 AM)
QUOTE (alex_osu3 @ Apr 14 2004, 08:06 AM)
Dear all,
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Alex
Hi Alex,
I'm having sort of the same problem as you currently. In my case however, my actin primers (positive control) are giving me the correct sized band in +RT and -RT reactions. I'll post again if I'm close to solving it. Likewise, I'd appreciate any help as well if you've got some suggestions. Perhaps we can help each other solve it this way.
This means you have contamination in your -RT reactions. There is DNA in one of your solutions, or is being introduced to your negative reactions through a contaminated pipettor, etc. I would start by cleaning everything, changing your water, and making new primer stocks. (Reorder your primers if you have to). If you have actin bands in your -RT, but no bands for your transcript of interest (assuming you got the transcript band in your +RT), then it's possible that your actin primers have been contaminated.
Hi MolBioGirl,
Didn't think I'd see you here. Anyway, I'm quite sure my actin primers are not contaminated because my negative control (no template, just water) turned out negative......thankfully. Also my +RT did not give any result for my transcript of interest. Anyway, I've posted the problem in my original topic "Difficulty synthesising cDNA..." in the Molecular Biology forum. See you there. Thanks.
-calavera1984-