Team:BCCS-Bristol/Protocols-Agarose Gel Electrophoresis

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Latest revision as of 20:40, 13 September 2008

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Agarose Gel Electrophoresis

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)
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
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

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-==Agarose gel electrophoresis==
+==Agarose Gel Electrophoresis==
 ===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)
+#	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: ===
--	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: ===
--	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
--	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
--	Use 5 µl HyperLadderI (BIOLINE)
+#	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
--	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.
+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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