I want to develop a HCV viral load determination based on quantitative real time pcr. I have used light cycler 480 and MGb probes and Ez kit. I have used a dilution of 1e7 up to 1e1 as a standard. I optimized the reaction successfully but when I repeated it again after 2-3 times I was confronted with problems. Now my problem is on my standards, the cross point of 1e3,1e2 and 1e1 are the same or so closed .
My mix per reaction includes:
water 0.5 ul
buffer 3 ul
Mn 25 mM 2.5 ul
dNTP 10mM 0.7ul
primers 10 pmol 0.6 ul
prome 5pmol 0.6 ul
UNG 1u/ul 0.1ul
Rth poly 2.5 U/ul 0.5ul
GAPDH primer probe 0.6ul
template 2.7 ul
real time steps
UNG 50 centigrade 2 min 1 cycle
revers trans 60 centigrade 30 min 1 cycle
denaturation 95 centigrade 5 min 1 cycle
Amplification 95 centigrade 20 sec
60 centigrade 1 min 40 cycle
cooling 40 centigrade 30 sec 1 cycle
Hi Husseini!
So I suggest you remove the three points on the line with the standard curve problem and then analyze the slope and R2 to verify that are appropriate. If getting, you can just remove them, because the program will calculate the absolute amount of viral load from the equation of the line generated. Moreover, in the same situation in the other five remaining points form a good standard curve, his error may have occurred in the sample dilution pipette uncalibrated ... ...
Another suggestion is to increase the amount of DNA in the DNA samples.Quantidade may have been insufficient for analysis. Then it will increase the amount of DNA in all the samples and repeat the curve again.
If does not work out, then you could move in reaction conditions (MIX, enzymes ...).
When I had this problem this way was guided by my advisor.
Hope this helps. If someone has other solutions will be very useful for me too.
Best Regards
Michella
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