I did two series of dilution. One dilution for G12R mutations is from 20k copies/uL to 2k copies/uL to 200copies/uL to 20copies/uL in ultrapure water.
The other dilution for G12R mutations is from 20k copies/uL to 2k copies/uL to 200copies/uL to 20copies/uL in constant WT solutions. I mixed these DNA samples with mastermix and make them amplified in the real-time machine. Then, I got weird Cq values. And the difference between WT and 20k/uL G12R is too small. The Cq value of WT is even smaller than that of 20k/uL G12R. Could you please tell me how can this situation happen? I am pretty sure that I have done the correct dilution because I made a planning sheet and wrote down every steps I should do. I tick the reagents after I added them into tubes. And I even changed gloves every time I need to pipette DNA samples. The WT and G12R are kept separately. They are not taken simultaneously into the biosafety hood.
Assuming you didn't make a math error, pipetting error, or mixing error, I'd say the most likely issues are:
- your primers aren't efficient enough (95-105% is necessary)
- One or more of your reagents is degraded (primers, DNA or Master Mix)
Your error bars are way, way too large. That tells me that either you have uncalibrated pipettes, poor pipetting skills, evaporation from your plates, or degraded reagents.
Lots of trouble-shooting for you. First step, double check your math. Then make sure your pipettes are calibrated. Then toss out ALL of your reagents and try again.
Good luck!
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