I am aiming to reveal genetic similarities among hybrids. Therefore, I made an SRAP analysis in tomato, pepper and melon. However, every time I observed a band in negative controls on agarose gel. The number and pattern of the bands were the same as that I observed in my sample. PCR reactions and primer combinations are below. Could you please help me how to get rid of the amplicons observed in negative control?
Primers Combinations
- Me1-Em2
- Me1-Em3
- Me2-Em12
- Me5-Em1
- Me2-Em8
PCR CYCLES
- 94 °C 5 min
- 94 °C 1 min- 40 °C 1 min- 72 °C 1 min 5 cycles
- 94 °C 1 min- 55 °C 1 min- 72 °C 1 min- 30 cycles
- 72 °C 10 min
- 4 °C ∞
Reaction Mixture for Samples
1X
Reaction Mixture for Negative Control
1X
Kamaran Salh Rasul I added ultra-pure water, primer pair and PCR master mix.
Kamaran Salh Rasul I thought that as you mentioned master mix or primer might be contaminated. However, all the primer pairs have produced a band in the negative control. So, it may not be about contamination issues. Additionally, when I used the master mix for screening breeding lines against different diseases, there were no bands in negative controls.
I agree with Kamran. Use these reactions and send the image. 1) change the Water. 2) change the Master Mix. 3) use e diffrent primer.
Please check that the instrumental used for PCR reaction are clean. The bands are clear, so you have a contamination. If you are sure that the water, the master mix and the primers are free of contaminants, maybe you can test the pipettes, microtubes, or any instrumental used that can have contaminants.
Thank you Mostafa Haghpanah and Kamaran Salh Rasul . I am checking every component of the PCR reaction that might be contaminated somehow.
I will also check instruments like pipet tips, and PCR tubes as María I. Dinolfo suggested. Thanks for your suggestion María I. Dinolfo
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