{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %} {% block page_content %}
Names: Danielle Dotson, Molly Ripper, Chambre Garcia, Matthew Gaston, Austin Thrasher
Start Time: 1:30 pm
Purpose: In Vitro transcription to produce LwCas13a-associated crRNA and miRNA-424-5p
(page 6)
Compoent | Amount |
---|---|
DEPC-treated water | to 20 µL → 5.5 µL |
5X TranscriptAid Reaction Buffer | 4 µL |
ATP/CTP/GTP/UTP mix* | 8 µLb |
Template DNA | 2 µg → 0.5 µL |
TranscriptAid Enzyme Mix | 2 µL |
Total volume: | 20 µL |
* Equal volumes of the four provided NTP solutions combined in one tube.
Tubes: (miRNA, trans, 4/20) and (crRNA, trans, 4/20)
We should have a tube of miRNA 424-5p and a tube of crRNA for the Cas13 enzyme.
Notes: None
Stop Time: 7:00 pm
Next Time: Purification of RNA transcripts.
Names: Molly Ripper, Chambre Garcia, Matthew Gaston, Austin Thrasher
Start Time: 7:30 pm
Purpose: RNA Purification of transcription products from the last experiment
The tubes appear to be more transparent than the original tubes, indicating that this RNA had some level of purification.
Notes: Since we were unsure if ethanol had already been added to the RA3 buffer from this kit, we substituted RNA wash buffer from the Zymo kit. We proceeded to run the miRNA on the nanodrop to determine the concentrations. The concentration of the miRNA is 9.8 ng/uL and the concentration of the crRNA is 25.6 ng/uL.
Stop Time: 8:30 pm
Next Time: Test the components in the full Cas13 system to achieve proof of concept.
Names: Erik Bergstrom, Danielle Dotson, Molly Ripper
Start Time: 2:30 PM
Purpose: To test our Cas13 system for initial functioning and proof of concept.
Component | Starting Concentration | Amount |
---|---|---|
Milli-Q Water | 3.5 ul | |
crRNA | 25.6 ng/ul | 0.5 ul |
Cas13 Enzyme | 63.3 ug/ul | 2 ul |
Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | 10 X | 2 ul |
miRNA | 9.8 ng/ul | 12 ul |
Total Volume: | 20 µL |
Two tubes contain our system. The ideal outcome is that both tubes contain fragments of miRNA that have been cut by Cas13.
Stop Time: 4:20 pm
Next Steps: Run a gel electrophoresis experiment using ethidium bromide to determine if the miRNA was cut in the presence of Cas13.
Names: Molly Ripper, Erik Bergstrom, Danielle Dotson
Start Time: 3:30 PM
Purpose: To run the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA.
Prepare a chart to keep track of which sample you will place in each well.
The gel only showed one band in the ladder lane. The three other lanes had no bands at all, but the loading dye could be seen.
Stop Time: 5:30 PM
Next Time: Repeating the experiment with a new method to detect cut miRNA.
Names: Erik Bergstrom
Start Time: 1:30 PM
Purpose: To test our Cas13 system for initial functioning and proof of concept.
Component | Starting Concentration | Amount |
---|---|---|
Milli-Q Water | 3.5 ul | |
crRNA | 25.6 ng/ul | 0.5 ul |
Cas13 Enzyme | 63.3 ug/ul | 2 ul |
Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | 10 X | 2 ul |
miRNA | 19.7 ng/ul | 12 ul |
Total Volume: | 20 µL |
Stop Time: 3:45 pm
Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.
Names: Erik Bergstrom
Start Time: 3:45 PM
Purpose: To run the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA.
Prepare a chart to keep track of which sample you will place in each well.
Stop Time: 5:30 pm
Next Steps: Running the gel with crRNA and miRNA combined as an additional control and a ladder would be a better validation of our protocol. Only one control lane was used in this experiment due to the team having insufficient miRNA for a second control.
Names: Erik Bergstrom
Start Time: 4:30 pm
Purpose: In Vitro transcription to produce miRNA-424-5p and other miRNA
Added MilliQ water to DNA from IDT for miR-16, miR-145, miR-10b, miR21 to suspend in water at 4 ug/uL
miR-16: 545 uL
miR-145: 525 uL
miR-10b: 605 uL
miR-21: 525 uL
Compoent | Amount |
---|---|
DEPC-treated water | to 20 µL → 5.5 µL |
5X TranscriptAid Reaction Buffer | 4 µL |
ATP/CTP/GTP/UTP mix* | 8 µLb |
Template DNA | 2 µg → 0.5 µL |
TranscriptAid Enzyme Mix | 2 µL |
Total volume: | 20 µL |
* Equal volumes of the four provided NTP solutions combined in one tube.
Tubes: miRNA 424, miRNA 424 10/8, miRNA 16, miRNA 145, miRNA 10b, miRNA 21
Stop Time: 9:00 PM
Several tubes of RNA were prepared that needed to be purified.
The concentrations of the RNA were:
Sample | Concentration (ng/uL) |
---|---|
miR-424-1 | 4.7 |
miR-424-2 | 5.1 |
miR-424-3 | 6.8 |
miR-424-4 | 6.6 |
miR-424-5 | 3.3 |
miR-424-6 | 4.1 |
miR-10b | 8.4 |
miR-16 | 7.9 |
miR-21 | 8.4 |
miR-145 | 6.2 |
Names: Danielle Dotson, Matthew Gaston, Chambre Garcia
Start Time: 6:00 pm
Purpose: RNA Purification of transcription products from the last experiment
Nanodrop results show a concentration of 22.7 ng/uL for miRNA-145, 28.4 ng/uL for miRNA 424, 12 ng/uL for miRNA-424 (10/8), 26.2 ng/uL for miRNA-16, 17.5 ng/uL for miRNA-21, and 6.2 ng/uL for miRNA-10b.
Notes: Since we were unsure if ethanol had already been added to the RA3 buffer from this kit, we substituted RNA wash buffer from the Zymo kit.
Stop Time: 8:30 pm
Next Time: Test the different miRNA with Cas13 to determine the specificity of the enzyme.
Names: Danielle Dotson, Molly Ripper
Start Time: 1:00 PM
Purpose: To test our Cas13 system for initial functioning and proof of concept using a more informative gel.
Component | Starting Concentration | Amount |
---|---|---|
Milli-Q Water | 6.5 ul | |
crRNA | 25.6 ng/ul | 0.5 ul |
Cas13 Enzyme | 63.3 ug/ul | 2 ul |
Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | 10 X | 2 ul |
miRNA | 28.4 ng/ul | 9 ul |
Total Volume: | 20 µL |
Label 7 tubes as shown below:
miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 |
---|
All tubes will have Milli-Q water and reaction buffer. The labels above indicate what will be added to each tube for the reaction.
Stop Time: 3:45 pm
Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.
Names: Molly Ripper, Danielle Dotson
Start Time: 1:30 PM
Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier.
Prepare a chart to keep track of which sample you will place in each well.
RNA Ladder | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
---|
Stop Time: 5:10 PM
Next Steps: Troubleshooting our gel.
Names: Molly Ripper, Danielle Dotson, Erik Bergstrom, Austin Thrasher
Start Time: 7:30 PM
Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier.
Prepare a chart to keep track of which sample you will place in each well.
RNA Ladder (5 ul) | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
---|
High contrast image
Low contrast image
Stop time: 9:00 PM
Next steps: Run the experiment again and attempt to visualize results on another gel. Try to denature the miRNA to ensure the miRNA runs on the gel properly.
Names: Erik Bergstrom, Star Nevded, Molly Ripper
Start Time: 11:30 AM
Purpose: To test our Cas13 system for initial functioning and proof of concept using a more informative gel. Also testing the effect of other miRNA’s on the system.
Component | Starting Concentration | Amount |
---|---|---|
Milli-Q Water | To 20 uL | |
crRNA | 25.6 ng/ul | 0.5 ul |
Cas13 Enzyme | 63.3 ug/ul | 2 ul |
Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | 10 X | 2 ul |
miRNA-424-5p | 28.4 ng/ul | 9 ul |
miRNA-10b | 8.4 ng/ul | 10 uL |
miRNA-21 | 8.4 ng/ul | 10 uL |
Total Volume: | 20 µL |
miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 | miRNA-10b + crRNA + CAS13 | miRNA-21 + crRNA + CAS13 | |
---|---|---|---|---|---|---|---|---|---|
miRNA | 9 | 0 | 0 | 9 | 9 | 0 | 9 | 10 | 10 |
crRNA | 0 | 0.5 | 0 | 0.5 | 0 | 0.5 | 0.5 | 0.5 | 0.5 |
CAS13 | 0 | 0 | 2 | 0 | 2 | 2 | 2 | 2 | 2 |
buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
water | 9 | 17.5 | 16 | 8.5 | 7 | 15.5 | 6.5 | 5.5 | 5.5 |
All tubes will have Milli-Q water and cas reaction buffer. The labels above indicate what will be added to each tube for the reaction.
Completed reactions ready to run on a gel
Stop time: 2:00 PM
Next steps: Running the solutions on a gel.
Names: Erik Bergstrom, Star Nevded
Start Time: 2:30 PM
Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier, and to test if RNA cutting, if any, is specific. Addition of a denature step.
Prepare a chart to keep track of which sample you will place in each well.
RNA Ladder (5 ul) | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
---|
First Set of Images
Second Set of Images
Stop time: 4:00 pm
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