Team:HKUST/Protocols/Agarose gel

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Revision as of 15:50, 26 August 2009

1. Agarose gel preparation and gel electrophoresis

Purpose: To check the result
Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker
- Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); Lamda DNA BstEII marker (highest band 8400bp, 1% argarose)
Procedure:
- To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE.
- Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to dissolve the agarose.
- Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL.
- Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
- Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
- Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.
- Add 1 μL loading dye per 5 μL of sample.
- Load the samples from left to right.
- Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
- Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
- Carefully remove the gel from the gel box and check the result under UV exposure.
Tips:
- Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
- Let bottom of the flask be immersed in a cup of cold water for faster cooling.
- In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.
Safety tips:
- Be sure to wear a glove before treating the hot flask.
- Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.

@@ Line 1: / Line 1: @@
 . Agarose gel preparation and gel electrophoresis
-  Purpose: To check the result
+*Purpose: To check the result
-  Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker
+*Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker
-  Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp);
+**Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); Lamda DNA BstEII marker (highest band 8400bp, 1% argarose)
-                     Lamda DNA BstEII marker (highest band 8400bp, 1% argarose)
+*Procedure:
-  Procedure:
+**To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE.
-  a.To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE.
+**Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to   dissolve the agarose.
-  b.Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to   dissolve the agarose.
+**Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL.
-  c.Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL.
+**Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
-  d.Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
+**Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
-  e.Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
+**Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.
-  f.Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.
+**Add 1 μL loading dye per 5 μL of sample.
-  g.Add 1 μL loading dye per 5 μL of sample.
+**Load the samples from left to right.
-  h.Load the samples from left to right.
+**Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
-  i.Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
+**Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
-  j.Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
+**Carefully remove the gel from the gel box and check the result under UV exposure.
-  k.Carefully remove the gel from the gel box and check the result under UV exposure.
+*Tips:
-  Tips:
+**Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
-  •Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
+**Let bottom of the flask be immersed in a cup of cold water for faster cooling.
-  •Let bottom of the flask be immersed in a cup of cold water for faster cooling.
+**In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.
-  •In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.
+*Safety tips:
-  Safety tips:
+**Be sure to wear a glove before treating the hot flask.
-  •Be sure to wear a glove before treating the hot flask.
+**Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.
-  •Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.