Date: 04/20/2023

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

Protocol:

(page 6)

Set-Up

Added 1125 µL of MilliQ water to 2023MissouriMinersT7CRRNA424 (crRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
Added 615 µL of MilliQ water to 2023MissouriMinersT7MIRNA424 (miRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
Keep TranscriptAid Enzyme Mix and nucleotides on ice.
Keep the 5X TranscriptAid Reaction Buffer at room temperature.
Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

Combine the following reaction components at room temperature in the order given:

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)

Results:

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.

Date: 04/20/2023

Names: Molly Ripper, Chambre Garcia, Matthew Gaston, Austin Thrasher

Start Time: 7:30 pm

Purpose: RNA Purification of transcription products from the last experiment

Protocol:

Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiments up to 100 µL.
In two 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load the lysate into the RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard the flowthrough and place the column back into the Collection Tube.
For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease-free Collection Tube
Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
Store the RNA at -20°C until ready to use

Results:

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.

Date: 9/24/23

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.

Protocol

Setup

Use literature to find the concentrations of components needed for the experiment, and use C1V1 = C2V2 to determine the amount needed.
Thaw components and divide both the miRNA and crRNA into separate 5 uL aliquots.
Dilute Cas13 enzyme to 63.3 ug/mL by adding 1 uL of the enzyme to 14.8 uL of storage buffer (comprised of 0.3 M NaCl 20 mM Tris-HCl, ph 8.0 0.5 mM EDTA 1 mM DTT 50% Glycerol)

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

Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to a small Eppendorf tube
Incubate the solution at 37 C for 30 minutes in a water bath.
Remove the tube from the water bath and add 2 uL of miRNA to the solution and mix carefully by gently inverting the tube.
Incubate the solution again at 37 C for 5 minutes.

Results:

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.

Date: 9/24/23

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.

Protocol:

Measure 0.5 g agarose powder and add it to a 250 mL flask.
Add 50 mL 1x TAE buffer to the flask.
Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
Carefully unscrew and lower the rails on the gel box.
Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. If the gel is placed in the wrong orientation, the DNA will run off the end of the gel and the sample will be lost!
Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
Carefully and slowly pull the comb straight up and out of the gel.
Prepare a chart to keep track of which sample you will place in each well.
1. Well 1 - Ladder
2. Well 2 - miRNA
3. Well 3 - Tube 3-1
4. Well 4 - Tube 3-2
Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
Add 10 μL of the ladder to an empty well.
Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
Run the gel immediately so that the RNA samples do not diffuse out of the wells.
Put the gel on a UV illuminator to see the results.

Results

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.

Date: 10/7/23

Names: Erik Bergstrom

Start Time: 1:30 PM

Purpose: To test our Cas13 system for initial functioning and proof of concept.

Protocol:

Setup:

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

Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to a small Eppendorf tube.
Incubate the solution at 37 C for 1 hour in a water bath.
Remove the tube from the water bath and add 2 uL of miRNA to the solution and mix carefully by gently inverting the tube.
Incubate the solution again at 37 C for 10 minutes.

Stop Time: 3:45 pm

Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.

Date: 10/7/23

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.

Protocol:

Measure 2 g agarose powder and add it to a 250 mL flask.
Add 50 mL 1x TAE buffer to the flask.
Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
Carefully unscrew and lower the rails on the gel box.
Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
Carefully and slowly pull the comb straight up and out of the gel.
Prepare a chart to keep track of which sample you will place in each well.
1. Well 1 - Reaction mixture from the previous experiment
2. Well 2 - miRNA (12 uL total miRNA)
Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
Run the gel immediately so that the RNA samples do not diffuse out of the wells.
Put the gel on a UV illuminator to see the results.

Results:

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.

Date: 10/8/2023

Names: Erik Bergstrom

Start Time: 4:30 pm

Purpose: In Vitro transcription to produce miRNA-424-5p and other miRNA

Protocol:

Set-Up

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
All tubes result in a concentration of 4 µg/µL in the original DNA tube.
Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
Keep TranscriptAid Enzyme Mix and nucleotides on ice.
Keep the 5X TranscriptAid Reaction Buffer at room temperature.
Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

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

Mix thoroughly, spin briefly to collect all drops, and incubate at 37°C for 4-8 hours (short transcript).

Stop Time: 9:00 PM

Results:

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

Date: 10/9/2023

Names: Danielle Dotson, Matthew Gaston, Chambre Garcia

Start Time: 6:00 pm

Purpose: RNA Purification of transcription products from the last experiment

Protocol:

Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiment up to 100 µL.
In six 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load lysate into RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard flowthrough and place the column back into the Collection Tube.
For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease free Collection Tube
Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
Store the RNA at -20°C until ready to use

Results:

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.

Date: 10/10/23

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.

Protocol:

Set-up

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.
Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
Incubate the solution at 37 C for 1 hour in a water bath.
Remove the tube from the water bath and add 9 uL of miRNA to the solution as well as enough Milli-Q to bring it to 20 uL and mix carefully by gently inverting the tube.
Incubate the solution again at 37 C for 10 minutes.

Stop Time: 3:45 pm

Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.

Date: 10/10/23

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.

Protocol:

Measure 2 g agarose powder and add it to a 250 mL flask.
Add 50 mL 1x TAE buffer to the flask.
Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
Carefully unscrew and lower the rails on the gel box.
Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
Carefully and slowly pull the comb straight up and out of the gel.
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
Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
Incubate in a 70°C water bath for 10 minutes.
Chill samples on ice for 3 minutes and spin briefly.
Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
Run the gel immediately so that the RNA samples do not diffuse out of the wells.
Put the gel on a UV illuminator to see the results.

Results

Stop Time: 5:10 PM

Next Steps: Troubleshooting our gel.

Date: 10/10/23

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.

Protocol:

Measure 2 g agarose powder and add it to a 250 mL flask.
Add 50 mL 1x TAE buffer to the flask.
Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
Carefully unscrew and lower the rails on the gel box.
Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
5 ul of ethidium bromide was added to the positive side of the electrophoresis tray to help avoid the EtBr running off of the gel.
Carefully and slowly pull the comb straight up and out of the gel.
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
Add 10 μL 2x RNA loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
Add 20 μL of each sample to a well using one sample per well, exhibiting caution.
Run the gel immediately so that the RNA samples do not diffuse out of the wells.
Put the gel on a UV illuminator to see the results.
The ethidium bromide had ran off of the gel again

Results:

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.

Date: 10/11/2023

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.

Protocol:

Set-up

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.

Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
Incubate the solution at 37 C for 1 hour using a PCR machine
Remove the tube from the water bath and add 9 uL of miRNA to the solution, mix by inverting the tube
Incubate the solution again at 37 C for 10 minutes.

Results:

Completed reactions ready to run on a gel

Stop time: 2:00 PM

Next steps: Running the solutions on a gel.

Date: 10/11/23

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.

Protocol:

Measure 2 g agarose powder and add it to a 250 mL flask.
Add 50 mL 1x TAE buffer to the flask.
Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
Carefully unscrew and lower the rails on the gel box.
Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
Carefully and slowly pull the comb straight up and out of the gel.
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
Add 10 μL 2x RNA loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
Incubate in a 70°C water bath for 10 minutes.
Chill samples on ice for 3 minutes and spin briefly.
5 ul of ethidium bromide was added to the positive side of the electrophoresis tray to help avoid the issue with the EtBr running off the negative end of the gel.
Add 20 μL of each sample to a well using one sample per well, exhibiting caution.
Run the gel immediately so that the RNA samples do not diffuse out of the wells.
Put the gel on a UV illuminator to see the results.
The gel was allowed to soak in TAE buffer with EtBr for 40 minutes after being imaged initially
The gel was imaged again on an UV illuminator

Results:

First Set of Images

Second Set of Images

Stop time: 4:00 pm