Hi, I am a masters student working on a PCR experiment. I do not have access to a spectrophotometer or a nano drop. Is RNA quantitation like this able to be done on a microplate reader?
Hello. I've been working on a project that requires me to put standards of several milk-derived amino acids into a UV spectrophotometer, but I can't seem to get any of them. I am unable to determine what wavelengths they absorb light because they all have a noise peak in the 200-300 nm range. I attempted adjusting the standards' concentrations, but to no avail. What do you think might be the issue?
How to use the nano-drop to estimate the concentration of a recombinant purified protein ? Abs(280) = Ext.coef x Conc. x path length. the path length is 1 mm for nano drops right ?? although on the machine it says 10 mm I dont know why ! more importantly, when I use the amino acid sequence to find out the estimated ext. coef. I get two values: 1)Ext. coefficient 27890 M-1 cm-1 and 2)Abs 0.1% (=1 g/l) 0.804 which extinction coef. to use and how can I solve the equation with these units. My protein conc. appears to have 10 mg/ml without making any calculations, so given tbis extinction coefficient and using the nano drop what would be the actual conc..I got really confued
The nanodrop typically adjusts the values to correspond to the 1 cm (10mm) length which is the standard. The Abs is not the extinction coefficient. Those are different but connected. The Abs 0.1% (=1g/L) is telling you that a concentration of 1g/L (1 mg/mL) will have an absorbance of 0.804. So you can divide your measured absorbance by that to figure out mg/mL.
It's giving you total protein concentration in mg/mL, so it doesn't matter what state the protein's in (or even what proteins there are) - it's only if you convert to molarity that that would come into play, but the assay can't tell you that
@@thebumblingbiochemist right, so I can still rely on it to calculate the protein conc. in mg/ml. Then I can convert them to molarity using the mol. Wt of the monomer, then I have the concentration of the sample as a monomer.
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this is gold.
I was looking for an answer why the equation has value 50 .... But i got the whole explanation .... Thank you so much 🌻
Whatever you can find cheap :) Just make sure it will do the wavelengths you want and you have compatible cuvettes (no glass or normal plastic for UV)
Thank you, you are extraordinary
Hi, I am a masters student working on a PCR experiment. I do not have access to a spectrophotometer or a nano drop. Is RNA quantitation like this able to be done on a microplate reader?
maybe check out this? www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorescence-microplate-assays/microplate-assays-nucleic-acids.html
Hello. I've been working on a project that requires me to put standards of several milk-derived amino acids into a UV spectrophotometer, but I can't seem to get any of them. I am unable to determine what wavelengths they absorb light because they all have a noise peak in the 200-300 nm range. I attempted adjusting the standards' concentrations, but to no avail. What do you think might be the issue?
What's your buffer?
Distilled water
And you don't see the peak in your distilled water blank?
How to use the nano-drop to estimate the concentration of a recombinant purified protein ?
Abs(280) = Ext.coef x Conc. x path length.
the path length is 1 mm for nano drops right ?? although on the machine it says 10 mm I dont know why !
more importantly, when I use the amino acid sequence to find out the estimated ext. coef. I get two values:
1)Ext. coefficient 27890 M-1 cm-1
and
2)Abs 0.1% (=1 g/l) 0.804
which extinction coef. to use and how can I solve the equation with these units.
My protein conc. appears to have 10 mg/ml without making any calculations, so given tbis extinction coefficient and using the nano drop what would be the actual conc..I got really confued
The nanodrop typically adjusts the values to correspond to the 1 cm (10mm) length which is the standard. The Abs is not the extinction coefficient. Those are different but connected. The Abs 0.1% (=1g/L) is telling you that a concentration of 1g/L (1 mg/mL) will have an absorbance of 0.804. So you can divide your measured absorbance by that to figure out mg/mL.
this was helpful, cheers!
Great to hear!
@@thebumblingbiochemist do you happen to have a video on calculating RNA yield?
not more than this one sorry
NICE VIDEO
Glad you found it helpful!
It helps me a lot thank u😊
Happy I could help!
thanks
happy to help
If the protein can form a dimer, then if I use bradford assay with (BSA standard), does it give me the concentration of the dimer or the monomer ?
It's giving you total protein concentration in mg/mL, so it doesn't matter what state the protein's in (or even what proteins there are) - it's only if you convert to molarity that that would come into play, but the assay can't tell you that
@@thebumblingbiochemist right, so I can still rely on it to calculate the protein conc. in mg/ml.
Then I can convert them to molarity using the mol. Wt of the monomer, then I have the concentration of the sample as a monomer.
Yes. But you won't know the actual oligomerization status
Nice
thanks!