Comments on Jake Heare Research Central: 5 5 2015 310 Heat Shock RNA Isolation / qPCR

Sorry, just one more thing I notice; I promise! Y...

2015-05-06T14:44:03.359-07:00

Sorry, just one more thing I notice; I promise!

Your centrifugation numbers are wrong. The g-force values you have listed don't match the RPMs (or, vice versa, but I think you were using the RPM setting on the centrifuge yesterday).

On the centrifuge you were using 7400RPM = 5143g. 11400RPM = 12205g.

I'd recommend just using the RCF (g) setting on the centrifuge.

There's really no reason to use (nor report) RPMs, since RPM values will have different corresponding RCFs across different centrifuges (RCFs are dependent upon the radius of the rotor in the centrifuge).

Oh, one more thing. I noticed that on your qPCR ru...

2015-05-06T09:49:20.935-07:00

Oh, one more thing. I noticed that on your qPCR run from yesterday (http://goo.gl/kqshoz) and this one that your melt curves did not complete. Did you happen to stop the program before it finished on both runs? The melt curve protocol should go to 95C, but on yesterday's it stops at ~69C and on this one it stops at ~71C.

Big picture questions regarding your results: If ...

2015-05-06T09:28:23.018-07:00

Big picture questions regarding your results:

If you still have gDNA contamination in your RNA, why would you proceed to making cDNA? If you make cDNA from these samples, how will you know if amplification produced in qPCRs run using the cDNA is due to residual gDNA or from the cDNA? Additionally, if you're OK with having contaminating gDNA in your RNA, why perform the DNasing in the first place?

Other things I noticed:

- Your qPCR reactions still don't add up to 20uL. Additionally, there's some discrepancy in your notebook as to what the final volume of the qPCR reactions actually is. The table shows a final volume of 20.3uL but in the text you say that you added 0.5uL to 20uL of master mix - final volume 20.5uL. As I mentioned previously, the degree of deviation from the proper final volume probably has no noticeable impact on your reactions, but you should correct it in future qPCRs.

- You're still using the term "negative control", but I think you mean "no template control." Your notebook also isn't clear on whether you substituted water for DNA template (which is necessary to have a proper no template control). An example of a negative control would be to use C. gigas DNA in a qPCR reaction instead of O. lurida DNA. A no template control uses water as template instead of any DNA.