Inconsistant curves - (May/12/2008 )
Dear all
I am not getting smooth and consistant curves in QPCR. I am frustrated can anyone tell me it could be due to my pipetting error? Besides I dont use filter tips during my master mix(MM) preparation. Could it be the reason? I guess not.
I normally prepare MM and distribute it to 0.2 ml tubes. Then put samples in them, briefly vortex them, spin them and run thermal cylers.. IS there any mistake?
Somebody help me out.
Sincerely
Phosphodiester
I am not getting smooth and consistant curves in QPCR. I am frustrated can anyone tell me it could be due to my pipetting error? Besides I dont use filter tips during my master mix(MM) preparation. Could it be the reason? I guess not.
I normally prepare MM and distribute it to 0.2 ml tubes. Then put samples in them, briefly vortex them, spin them and run thermal cylers.. IS there any mistake?
Somebody help me out.
Sincerely
Phosphodiester
Pipetting error may just give you higher or lower Ct value, it should not affect teh curve quality as long as it is within linear range.
So many things have to be right in order to get consistent curves! A small list here..
1. Unexpired, properly stored MM.
2. Amount of template DNA you put.
3. Quality of template DNA, PCR inhibitors etc.
4. Right PCR conditions, tm, proper primer design, intron-spanning if it is cDNA qPCR etc,.
You just have to be absolutely sure of all steps, all reagents, all design and do them right to get consistent results. And do a control qpcr that works in other people's hands to see if you can make it work with all your techniques. It may take time, but not so much in the long run!
Dear cell counter,
Thanx for your suggestions. you are right about appropriate storage... I was having trouble with my freezer in the lab.. any way thanx..
Dear cell counter,
my earlier query was for HIV detection. Later i used a kit that for M. laprae diagnosis. Template isolation was done from infected mouse. This time my kit contained inhibition control too. I used both FAM and JOE as reporter dye. My curves were not consistent this time also. Curves with JOE (for inhibition) were not sigmoidal at all. there were signals in all of the standards and sample but not to the appropriate shape.
Samples showed highly positive with FAM dye. I could not interpret my results. I am afraid if it was due to my inaccuracy or lack of skill. Please suggest me or refer any article that gives characteristic of inhibition control curves.
sincerely