I ahve problem with 20ul final reaction volume. my Positive control works and i have a good amplification curve but my DNA samples do not work! is there any fixed reaction volume that has worked good enough for all experiments?
20ul reaction that I used:
2 ul DNA sample
10 ul SYBR green
0.4 ul primer (each)
7.2 ul water
I feel 2ul DNA may be too much,
I would be thankful if you could tell me your opinion?
Hello Mandana, I know in our lab we used to use the 20 ul as the final volume but then we changed it to 10 ul:
5 ul SYBR green
0.5 ul each primer
1.5 ul water
2.5 ul cDNA
and it works perfectly in our hands.
You can try this, all our primers are designed for 61 C annealing temperature.
Hi Mandana, 10 or 20ul reactions should not really matter (except financially).
I would start with 1 ng gDNA (for a mammalian genome size) in a 10ul qPCR reaction. If you suspect potential inhibition do a 1/4 dilution and check the sinusoidal curve. If the high concentration curve have a lower maximal slope and/or plateau at a lower fluorescence it is most likely that you got inhibition in the reaction which can be diluted out.
For the Primer I would use 0.3 uM final amount in the 10ul reaction with higher concentration you more easily get artifacts.
Mix well, no emulsion, no air bubbles in the well bottom
Hope this helps
Hannes
Hi Mandana Askarizadeh , I used this protocol and got satisfactory results.
SYBR Green Master Mix (10 μl)
RNase-DNase free Water (5.6 μl)
Forward Primer (1.2 μl / 0.2 μM)
Reverse Primer (1.2 μl / 0.2 μM)
cDNA (2 μl / 100 ng/μl)
Total: 20 μl
Hannes Richter Thanks, Hannes for your answer!
I would ask you about how you mix your reactions in wells while SYBR green is easy to make bubbles when pipetting? I try to get ride of bubbles with centrifuge but it doesn't work sometimes!
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