New answers tagged pcr
Realtime PCR uses a fluorescent dye which binds to double-stranded DNA and thus allows to measure the growing amount of DNA by each cycle. The DNA added to the reaction also binds the fluorescent dye and makes some part of the fluorescent background. When the PCR reaction starts, it takes a while until enough DNA is synthesized to go over the background and ...
What I can quickly think of is that you were running the gel incorrectly: instead of from - to + direction, You ran it the other way around and your DNA went out of the gel. Another thing might be that the UV light was not turned on or is broken.
You have to scale amounts of all PCR components proportionally depending on the volume of the reaction. The polymerase is no exception in this respect, the only difference is that you measure it in terms of activity - units. So in your protocol you have 0.025 U/µl and you have to maintain this concentration when changing the volume.
The explanation I got when learning how to do qPCR was that you need run both replicate experiments and parallel experiments to get publishable data. Replicates (or biological replicates) would be qPCR runs on independently collected samples. That does not count your control vs your experimental groups. So for my work I had to collect 3 sets of control ...
How many cycles of PCR before dNTPs run out? Assume a 25 μl reaction. Assume 200 μM dNTPs. 200 μM dNTPs = 200 pmol μl -1 so in 25 μl reaction, there are 5000 pmol of dNTPs = 5000 x 10-12 x 6 x 1023 molecules = 3 x 1015 molecules dNTP Assume that we start with 1 molecule of a 1000 bp template, 50% GC 1 kb = 2000 nucleotides So , ...
I would draw the line beyond 35, but thats a bit cosmetic. The reasons are manyfold: due to the exponential fashion of the amplification (ideally) reagents are used up at some point reagents degrade, this is especially true for the dNTPs the activity of the enzyme, despite being heat-stable is declining over time beyond 35 cycles the exponential curve is ...
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