4 votes
Accepted

What is the minimum Ct value that you would consider indicates no gene expression?

Can't you just do a negative control and use that to calibrate? Alternately, run the DNA from your PCR on a gel with a positive control indicating expression. Literally zero expression should mean ...
Maximilian Press's user avatar
2 votes
Accepted

Explanation of QPCR's "mixing curves."

Probably easier to help diagnose a problem if you upload a screen shot of the amplification curves and/or melt curves. One issue that a lot of people run into when starting out is that the dilution ...
MikeyC's user avatar
  • 4,714
2 votes

How to compare basal expression of a gene in two tissues using qPCR techniques?

You normalized the data to the control and obtained the normalized ct-values. Let's call it Δct. Now you need to find the relative difference between them. In order to do that, the first step is that ...
Felix H.'s user avatar
  • 318
2 votes

PCR: Are primers designed to be complementary to a DNA sequence within the template DNA or to the 3' ends of the template DNA?

You seem to be confused about the template DNA having a relevant "end". Most DNA molecules one would start with would be very long, often an entire chromosome. The 3' ends of the two ...
mgkrebbs's user avatar
  • 9,054
1 vote
Accepted

Qpcr Copy number formula

The second formula (2^-ΔΔCt) you are presenting does not calculate a number of copies of your target. It is instead used in relative quantitation, where you want to know the relative expression of ...
CaroZ's user avatar
  • 414
1 vote
Accepted

modify PCR steps to include a ligation

In case this helps anyone, I found a technique called Gap Ligase Chain Reaction which includes both a thermostable ligase and a DNA polymerase enzyme in the same reaction: Ligase Chain Reaction (LCR) -...
Ryan's user avatar
  • 121
1 vote

What should be considered a GC clamp in a qPCR primer?

I have never heard it being called a "GC clamp" but designing primers with GC near the 3' end is a standard way to make the 3' end duplex more stable, allowing for greater efficiency but ...
mmhryc's user avatar
  • 11

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