NanoBio: AgaroseGels

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===How fast will my gel run?===
===How fast will my gel run?===
-
Higher voltage and your DNA will migrate faster. It will also heat up the buffer⋛ don't melt it!
+
Higher voltage and your DNA will migrate faster. It will also heat up the buffer and gel, and bands may appear diffuse.
(Please add your own results too!)
(Please add your own results too!)
-
*1% agarose 50mL with Qiagen GelPilot 5x - orange <100bp band hits bottom ~20 minutes at 150V
+
*1% agarose 50mL: ~20 minutes at 150V
 +
*1% agarose 40mL: ~12 minutes at 222V
==References and Links==
==References and Links==

Revision as of 13:20, 23 January 2009

Contents

Introduction

Agarose gels help you visualize DNA. Cool!

You can make agarose from 0.5% to even 3%, by mass. 0.7% shows separation of large fragments (5-10kb) and 2% shows good separation of small fragments (0.2k-1k). Keep in mind the gelliness/solidness positively correlates with more agarose; 0.5% gel will be floppy and fragile.

Agarose % guide

Agarose (g/100mL) DNA resolution (/kb)
0.5 1-30
0.7 0.8-12
1.0 0.5-10
1.2 0.4-7
1.5 0.2-3

Taken from Agarose gel electrophoresis.

Ingredients

  • TAE or TBE, 1x - 50 mL is good for a little gel but you have to determine empirically
    • Don't run a TAE gel in TBE
    • Add extra volume of TAE to compensate for boiling
    • If gel purifying: add 1 mmol/L guanosine to the TAE/TBE. This does not affect ligation and other downstream steps, however it will protect sticky ends. (See references)
  • 0.5-2% agarose, by mass (e.g. for 1%, dissolve 0.5g agarose in 50 mL)
  • Ethidium Bromide (for 10mg/mL, use 1uL per 50mL TAE)

Procedure

  1. Pour TAE into a flask that you can swirl to mix the contents
  2. Add agarose and swirl vigorously. It will be cloudy
  3. Microwave until it boils. Take it out and swirl (caution it's hot). Keep microwaving until the agarose flakes are minimized.
  4. Let it cool somewhere until around 50 C - to minimize EtBr vapours
  5. Add EtBr and swirl
  6. Pour into a gel tray and let it solidify
  7. Load samples and run gel.

How fast will my gel run?

Higher voltage and your DNA will migrate faster. It will also heat up the buffer and gel, and bands may appear diffuse.

(Please add your own results too!)

  • 1% agarose 50mL: ~20 minutes at 150V
  • 1% agarose 40mL: ~12 minutes at 222V

References and Links


Arthur K Yu 20:45, 17 June 2008 (UTC)

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