Bitan:Calculating the yield and specific activity of labeled RNA

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Calculation of specific activity and yield of RNA
CTP used was from Perkin Elmer CTP, α-32P -3000 Ci/mmol, 10 mCi/ml, 250 µCi. Reference date for this lot was 11/21/09 so the activity is calculated as following: The activity of α-32P-CTP, lot RSO0117175 was 0.748 of total activity today based on the reference date of 11/21/09 and according to the decay chart. This is 6 days old. According to this formula, the total activity was calculated to be 2447 Ci/mmol:
Image:Untitled_2.png
where SAtheo.=9120 for the theoretical specific activity of carrier-free 32P; SAcal.=specific activity after the calibration date; and DF is the fraction of current radioactivity that will remain on the calibration date from the Decay Chart. So in this case
Image:Untitled_3.png.
After the reference date, the total activity of the fresh hot CTP was 250 μCi in 25 μl, so 10 μCi/μl (10 mCi/ml). But now, it is 187 μCi because 0.748×250=187. For example if 4 μl was used for in-vitro transcription, the total amount used would be
Image:Untitled_4.png
for each RNA pool. Hence the amount of 32P-CTP used would be
Image:Untitled_5.png.
The amount of cold CTP used was
Image:Untitled_6.png;
the total amount of CTP (cold and hot) is 1012.3 pmol because 1 μl cold CTP which is 1000 pmol was used. The molecular mass of RNA
Image:Untitled_7.png
(where 321.455g/mol is the average mass of rNTPs, 100 nt is the number of bases, 17 nt is the number of bases in the T7 promoter which are not transcribed, 61.96 is a constant for net difference due to missing 5’ phosphates and additional 5’ and 3’ hydrogen). If all the CTP were incorporated, the theoretical mass of RNA would have been 1012.3 pmol×26618.8 pg/pmol= 26.9 μg.
Before G-50 purification: 560684 cpm/μl
After G-50 purification: 437010 cpm/μl
Hence percent incorporation: 78% i.e.,
Image:Untitled_1.png
Because incorporation has been 78% for this RNA pool, then the mass of RNA produced is 21 μg. So the specific activity would be
Image:Untitled_8.png
and in terms of moles,
Image:Untitled_9.png.
To get 66 pmol RNA one needs 4.1 μl of RNA product after G-50 purification.
Also look at the Ambion Specific-Activity Calculator.
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