Mike Barnkob:Protocols/Cloning/Gels

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Creating and running gels

Gel protocol for dummies.

Reagents

1x TBE buffer:

• Dilute 100ml of 10x TBE buffer into 900 ml of dH20.

Reagents and instruments:

• Agarose
• SYBR safe gel stain
• DNA ladder
• Electrophoresis tank with plates

Protocol

To create 55 ml of 1% agarose buffer:

1. Add 0.55g of agarose to 55 ml of 1x TBE buffer.
2. Swirl and heat for 2-4 minutes in microwave.
   Note: It is a good idea to microwave for 30-45sec, stop and swirl, and then continue towards a boil.
3. Let solution cool to around 50 °C and add 5 μL of SYBR Safe gel stain.
   Note: Presumable more safe the ETBr, but anything that binds DNA should be treated as a carcinogen.
4. Pour in plate and let rest for 1 hour.
   Note: Or place in 4°C for 15-20 min.

To run:

1. Mix 10 μL of sample with 5 μL of loading dye
2. Transfer 15 μL to gel in electrophoresis tank and cover gel in TBE buffer.
3. Run for around 60 min at 80-150V V.
   Note: The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.
   Note: Check the run by looking at the band - stop when it's around 2/3 down.

To analyze:

1. Visualize gel on ?

References

• https://www.addgene.org/plasmid-protocols/gel-electrophoresis/