Team:BCCS-Bristol/Protocols-Agarose Gel Electrophoresis

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==Agarose gel electrophoresis==
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==Agarose Gel Electrophoresis==
===Gel preparation:===
===Gel preparation:===
-
- 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
-
 
+
# Pour the gel into a chamber with a comb that you prepared before
-
- Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles
+
# Let the gel cool down and become solid (15-20 min)
-
 
+
-
- Pour the gel into a chamber with a comb that you prepared before
+
-
 
+
-
- Let the gel cool down and become solid (15-20 min)
+
===Sample preparation: ===
===Sample preparation: ===
-
- Add 10 % “Sample Loading Buffer” (BIORAD) to the sample
+
# Add 10 % “Sample Loading Buffer” (BIORAD) to the sample
-
 
+
# Mix gently and spin shortly down
-
- Mix gently and spin shortly down
+
===Loading the gel: ===
===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!!!)
+
# 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
-
- Fill the chamber with 0.5x TBE until the gel is covered
 
-
- Use 5 µl HyperLadderI (BIOLINE)
+
The Sample Loading Buffer contains two dyes: Bromophenol blue runs at ~300 bp and Xylene cyanol FF runs at ~4 kb.
-
 
+
-
- 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.
+
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Latest revision as of 20:40, 13 September 2008

Home The Team The Project Submitted Parts Modelling Wet Lab Calendar Miscellaneous


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)
  2. Cool the solution until you can touch it with your hands for a longer time
  3. Add 0.5 µl ethidium bromide per 10 ml TBE and mix while avoiding air bubbles
  4. Pour the gel into a chamber with a comb that you prepared before
  5. Let the gel cool down and become solid (15-20 min)

Sample preparation:

  1. Add 10 % “Sample Loading Buffer” (BIORAD) to the sample
  2. Mix gently and spin shortly down

Loading the gel:

  1. 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!!!)
  2. Fill the chamber with 0.5x TBE until the gel is covered
  3. Use 5 µl HyperLadderI (BIOLINE)
  4. For colony PCR, put 5 µl of each reaction in the well
  5. Close the chamber with the lid (black pole to black=negatively charged and red to red=positively charged…)
  6. 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


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