--------------------------------------------------------------------------- KeyError Traceback (most recent call last) Cell In[2], line 24 21 os.environ['is_docker'] = 'False' 22 os.environ['repo_root'] = "/home/pranav/work/pranavmishra90/research-reference" ---> 24 os.chdir(os.environ['repo_root']) 26 # Load the subject's nanodrop readings 28 subject_nanodrop = pd.read_csv(f'protocols/molecular-biology/PCR/demo_nanodrop.csv') File <frozen os>:709, in _Environ.__getitem__(self, key) 706 value = self._data[self.encodekey(key)] 707 except KeyError: 708 # raise KeyError with the original key value --> 709 raise KeyError(key) from None 710 return self.decodevalue(value) KeyError: 'repo_root'
mRNA Isolation
Isolating mRNA from surgical samples
Protocol
Lyse samples and separate phases
- Add \(0.5\ mL\) of TRIzol into an Eppendorf tube containing each sample
- Transfer the contents to a mortar. Add \(50\ mL\) of liquid nitrogen (\(LN_2\)) and grind the sample. Repeat with additional \(LN_2\), as needed, grinding the sample into a fine powder.
- Transfer the Sample + Trizol powder into a fresh Eppendorf.
- Add \(0.3\ mL\) of TRIzol into each Eppendorf.
- Pipette mix to homogenize
- Incubate for 5 min for complete dissociation of the nucleoprotein complex
- \(0.25\ mL\) of chloroform per \(1\ mL\) of Trizol
- Incubate for 2-3 min
- Centrifuge the samples for 15 min at \(12,000g\) at \(4^{\circ}C\)
- Transfer the clear upper phase containing RNA to a fresh Eppendorf tube. Ensure you do not contaminate the aqueous phase by touching the whitish, DNA containing interphase.
- If desired, you can store the remainder of Trizol phase containing DNA and Protein at \(-80^{\circ}C\)
RNA Isolation
- Add \(0.5\ mL\) of Isopropanol to the aqueous phase per 1 mL of Trizol used
- Incubate for 10 min
- Centrifuge the samples for 15 min at \(12,000g\) at \(4^{\circ}C\)
- Visualize the total RNA precipitate at the bottom of the tube (whitish, gel-like pellet)
- Discard the supernatant with a pipette
- Wash pellet with \(1\ mL\) of cold (\(4^{\circ}C\)) 75% Ethanol per 1mL of Trizol used
- Vortex briefly
- Centrifuge the samples for 5 min at \(7,500g\) at \(4^{\circ}C\)
- Discard the supernatant by inverting the tube in one fluid motion, being careful to not disturb the pellet
- Use a vacuum centrifuge to dry the remaining amount of supernatant (~ 5 min)
- Resuspend the pellet in \(32\ \mu L\) of RNAse-free water (molecular grade water)
- Pipette mix to ensure complete resuspension
Determine the RNA Yield: Nanodrop
- Configure the nanodrop to export to USB CSV
- Pipette \(1.5\ \mu L\) of each sample into the nanodrop to measure the concentration
- Save the sample, giving it a name ##### RNA to USB CSV (##### is the 5 digit randomized study subject identification number)
- Paste the nanodrop readings into the corresponding CSV file / Excel file
--------------------------------------------------------------------------- NameError Traceback (most recent call last) Cell In[3], line 1 ----> 1 Markdown(tabulate(subject_nanodrop[['Date', 'Time', 'Sample Number', 'A230', 'A260', 'A280', 'A320', 2 'A260/A230', 'A260/A280', 'Concentration', 'Sample Name']], headers=['Date', 'Time', 'Sample Number', 'Sample Name', 'A230', 'A260', 'A280', 'A320', 'A260/A230', 'A260/A280', 'Concentration'],showindex="never", numalign='center', stralign='center', tablefmt="grid")) NameError: name 'subject_nanodrop' is not defined
Conversion to cDNA
--------------------------------------------------------------------------- NameError Traceback (most recent call last) Cell In[4], line 1 ----> 1 lowest_rna_concentration = min(subject_nanodrop['Concentration']) 3 rna_to_be_used = target_total_rna_ng_ul / lowest_rna_concentration 5 cdna_conversion = subject_nanodrop NameError: name 'subject_nanodrop' is not defined
<>:1: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
<>:3: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
<>:1: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
<>:3: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
/tmp/ipykernel_2875/3255309191.py:1: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
display(Markdown(f" Our target concentration of cDNA is {target_total_rna_ng_ul} $\\frac{{ng}}{{\mu L}}$."))
/tmp/ipykernel_2875/3255309191.py:3: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
display(Markdown(f"The least concentration of any sample is {lowest_rna_concentration} $\\frac{{ng}}{{\mu L}}$. To obtain the target concentration, we will need to use {round(rna_to_be_used, 2)} $\\mu L$ of the RNA previously isolated."))Our target concentration of cDNA is 1500 \(\frac{ng}{\mu L}\).
/tmp/ipykernel_2875/3255309191.py:1: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
display(Markdown(f" Our target concentration of cDNA is {target_total_rna_ng_ul} $\\frac{{ng}}{{\mu L}}$."))
/tmp/ipykernel_2875/3255309191.py:3: SyntaxWarning: "\m" is an invalid escape sequence. Such sequences will not work in the future. Did you mean "\\m"? A raw string is also an option.
display(Markdown(f"The least concentration of any sample is {lowest_rna_concentration} $\\frac{{ng}}{{\mu L}}$. To obtain the target concentration, we will need to use {round(rna_to_be_used, 2)} $\\mu L$ of the RNA previously isolated."))
```{=html}
————————————————————————— NameError Traceback (most recent call last) Cell In[5], line 3 1 display(Markdown(f“ Our target concentration of cDNA is {target_total_rna_ng_ul} $\frac