Team:BCCS-Bristol/Protocols-Agarose Gel Electrophoresis

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(Difference between revisions)
(Agarose gel electrophoresis)
(Gel preparation:)
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===Gel preparation:===
===Gel preparation:===
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- For a thick gel in a small chamber, boil 50 ml 0.5x TBE in the microwave (for a thinner gel to insert only 5 µl sample in each well 40 ml are sufficient)
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# For a thick gel in a small chamber, boil 50 ml 0.5x TBE in the microwave (for a thinner gel to insert only 5 µl sample in each well 40 ml are sufficient)
-
- Cool the solution until you can touch it with your hands for a longer time
+
# Cool the solution until you can touch it with your hands for a longer time
-
- Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles
+
# Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles
-
- Pour the gel into a chamber with a comb that you prepared before
+
# Pour the gel into a chamber with a comb that you prepared before
-
- Let the gel cool down and become solid (15-20 min)
+
# Let the gel cool down and become solid (15-20 min)
===Sample preparation: ===
===Sample preparation: ===

Revision as of 19:51, 12 August 2008

Agarose Gel Electrophoresis

Gel preparation:

  1. For a thick gel in a small chamber, boil 50 ml 0.5x TBE in the microwave (for a thinner gel to insert only 5 µl sample in each well 40 ml are sufficient)
  1. Cool the solution until you can touch it with your hands for a longer time
  1. Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles
  1. Pour the gel into a chamber with a comb that you prepared before
  1. Let the gel cool down and become solid (15-20 min)

Sample preparation:

- Add 10 % “Sample Loading Buffer” (BIORAD) to the sample

- Mix gently and spin shortly down

Loading the gel:

- Remove the comb and put the gel with the slide into a chamber (the wells need to be on the end with the black pole!!!)

- Fill the chamber with 0.5x TBE until the gel is covered

- Use 5 µl HyperLadderI (BIOLINE)

- For colony PCR, put 5 µl of each reaction in the well

- Close the chamber with the lid (black pole to black=negatively charged and red to red=positively charged…)

- Run the gel with 80-100 V


The Sample Loading Buffer contains two dyes: Bromophenol blue runs at ~300 bp and Xylene cyanol FF runs at ~4 kb.

BCCS-080812-HyperLadderI for electrophoresis.PNG