Ghost contamination in Real Time PCR - (Sep/12/2008 )
Hello everybody, I am struggling from last 3 months to overcome the contamination in my Real Time Experiments.
By using Taqman Probe, first I used Standard Curve to qunatify my sample, everything was OK.
I did my experiment on this basis and got good results, BUT
After two months I repeated the same experiment with same conditions ( reagents, place, well plate, tubes, tips, water), my negative control (template was replaced by autoclaved water) was Positive. I could not succeed to find the problem.
This time making another experiment trial with different primers, this time SYBR green, again I found that my negative control is positive. I took new primers for candidate and as well as for HKG, new autoclaved sterilzed tubes, new tips, pippete was washed throughly with Ethanol and tried best to avoid any contmination. The result was same.
I made 25 µl reaction for Sample (candidate) for each well and made duplicate and loaded 23µl in each well , for example in Row A, Sample 1 was loaded, 23µl in A1 and 23µl in A2 and Sample 2 was loaded in same way in A3 and A4 and I made three negative controls, A5 & A6, A7& A8 and A9 & A10.
GAPDH was used as HKG and were loaded in same way in Row B.
I am surprised to see the results that well A5 (Negative control of Sample) showed curve and C(t) value was 37.4 started after 36 Cycles and A6 to A10 all are like negative ( no curve, Ct value is zero). In HKG GAPDH well B6 had same result as A5 but all others B5, B7-10 were negative.
I realy donot where is the problem, my co-workers have not this problem. I failed to find the Ghost where from it comes and contaminate. Any suggestions!!!!!
By using Taqman Probe, first I used Standard Curve to qunatify my sample, everything was OK.
I did my experiment on this basis and got good results, BUT
After two months I repeated the same experiment with same conditions ( reagents, place, well plate, tubes, tips, water), my negative control (template was replaced by autoclaved water) was Positive. I could not succeed to find the problem.
This time making another experiment trial with different primers, this time SYBR green, again I found that my negative control is positive. I took new primers for candidate and as well as for HKG, new autoclaved sterilzed tubes, new tips, pippete was washed throughly with Ethanol and tried best to avoid any contmination. The result was same.
I made 25 µl reaction for Sample (candidate) for each well and made duplicate and loaded 23µl in each well , for example in Row A, Sample 1 was loaded, 23µl in A1 and 23µl in A2 and Sample 2 was loaded in same way in A3 and A4 and I made three negative controls, A5 & A6, A7& A8 and A9 & A10.
GAPDH was used as HKG and were loaded in same way in Row B.
I am surprised to see the results that well A5 (Negative control of Sample) showed curve and C(t) value was 37.4 started after 36 Cycles and A6 to A10 all are like negative ( no curve, Ct value is zero). In HKG GAPDH well B6 had same result as A5 but all others B5, B7-10 were negative.
I realy donot where is the problem, my co-workers have not this problem. I failed to find the Ghost where from it comes and contaminate. Any suggestions!!!!!
what instrument are you using?
I wouldn't suggest ABI or BioRad for real-time applications.......Rotorgene 6000 from Corbet is so reliable, so is LightCycler 2.0 from Roche.
I never trusted plate based real time assays. I would still go for microtubes or capillaries.
I wouldn't suggest ABI or BioRad for real-time applications.......Rotorgene 6000 from Corbet is so reliable, so is LightCycler 2.0 from Roche.
I never trusted plate based real time assays. I would still go for microtubes or capillaries.
I see your point, but how can you imply that AB platforms are unreliable?
I saw it before...
So, the way I got it is that you are using SYBR chemistry now? Have you performed the melting curves as well?
I am using BioRad cycler, 96 wells from same company. I thought earlier about 96 well, which prehaps might be contaminated, and are also replaced by AB, but no success.