Aug 01, 2023

Agarose gel for RNA and DNA

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo3bm7v4o/v1
  • 1University College of Dublin
kaylee Beine
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DOI: https://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
Keywords: agarose gels for rna, running agarose gel, dna sample, dna general method, rna, dna
Abstract
General method for running agarose gels for RNA and DNA samples
Protocol materials
TAE (Tris-Acetate-EDTA) buffer, 1x
Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML
RNA Loading Dye (2x) - 4.0 mlNew England BiolabsCatalog #B0363S
RiboRuler High Range RNA LadderThermo FisherCatalog #SM1821
SyberSafe DNA Gel StainInvitrogen - Thermo FisherCatalog #S33101
GeneRuler 1 kb Plus DNA LadderThermo FisherCatalog #SM1331
Preparing the agarose gel and plate
4m 30s
1 g agarose in 100 mL of TAE (Tris-Acetate-EDTA) buffer, 1x

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

4m 30s
Allow to cool before adding 1 µL Ethidium 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
Add 5 µL Ethidium bromide, 10mg/mL, 10mLAmrescoCatalog #X328-10ML in the gel medium within the chromatography chamber

Preparing RNA samples
5m
250-300 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 RNA Loading Dye (2x) - 4.0 mlNew England BiolabsCatalog #B0363S to each sample

Heat up the ladder RiboRuler High Range RNA LadderThermo FisherCatalog #SM1821 and RNA samples to 70 °C for 00:05:00 for the gel

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

Setting up the sample gels
1h 50m
Add 5 µL of ladder and samples to gel wells

For RNA: 80V for 01:00:00
For DNA: 80V for 00:40:00

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

10m