Aug 01, 2023

Public workspaceAgarose gel for RNA and DNA

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3bm7v4o/v1
  • 1University College of Dublin
kaylee Beine
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo3bm7v4o/v1
Protocol Citation: kaylee Beine 2023. Agarose gel for RNA and DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3bm7v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2023
Last Modified: August 01, 2023
Protocol Integer ID: 85764
Abstract
General method for running agarose gels for RNA and DNA samples
Protocol materials
ReagentGeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331
Step 7
ReagentTAE (Tris-Acetate-EDTA) buffer, 1x
Step 1
ReagentEthidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
In 2 steps
ReagentRNA Loading Dye (2x) - 4.0 mlNew England BiolabsCatalog #B0363S
Step 5
ReagentRiboRuler High Range RNA LadderThermo FisherCatalog #SM1821
Step 6
ReagentSyberSafe DNA Gel StainInvitrogen - Thermo FisherCatalog #S33101
Step 7
Preparing the agarose gel and plate
Preparing the agarose gel and plate
4m 30s
4m 30s
Amount1 g agarose in Amount100 mL of ReagentTAE (Tris-Acetate-EDTA) buffer, 1xContributed by users

Microwave solution in a flask for Duration00:03:00 until the agarose is dissolved. Microwave for Duration00:01:00 thereafter Duration00:00:30

4m 30s
Allow to cool before adding Amount1 µL ReagentEthidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML in the gel medium before completely cool. Then pour into the gel mold

Make sure that your plate is balanced and the teeth are in. Decide if you need a long ladder or a short weather.
Preparing the chamber
Preparing the chamber
Add Amount5 µL ReagentEthidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML in the gel medium within the chromatography chamber

Preparing RNA samples
Preparing RNA samples
5m
5m
Amount250-300 ng /RNA per sample C1.V1 = C2.V2

V1 = (10)(50)/RNA conc. (from nanodrop)

Add the V1 sample of RNA to 1:1 ratio of ReagentRNA Loading Dye (2x) - 4.0 mlNew England BiolabsCatalog #B0363S to each sample

Heat up the ladder ReagentRiboRuler High Range RNA LadderThermo FisherCatalog #SM1821 and RNA samples to Temperature70 °C for Duration00:05:00 for the gel

5m
Preparing DNA samples
Preparing DNA samples
ReagentSyberSafe DNA Gel StainInvitrogen - Thermo FisherCatalog #S33101 is already in the samples for the PCR; then use the ReagentGeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331

Setting up the sample gels
Setting up the sample gels
1h 50m
1h 50m
Add Amount5 µL of ladder and samples to gel wells

For RNA: 80V for Duration01:00:00
For DNA: 80V for Duration00:40:00

1h 40m
*Note: can check gel after Duration00:10:00 to see if there is product showing on the gel

10m