"
Team:Montreal/DNA Agarose Gel
From 2008.igem.org
m (→Gel Loading) |
|||
| (5 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
| + | {|style="font color="#ffffff"; background-color:#cd0000; cellpadding="3" cellspacing="5" border="2" bordercolor="#cd0000"border-spacing:6px; text-align:center" width="960px" | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal|<font color="#ffffff">Home</font>]] | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal/Team|<font color="#ffffff">The Team</font>]] | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal/Project|<font color="#ffffff">The Project</font>]] | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal/Parts|<font color="#ffffff">Parts Submitted to the Registry</font>]] | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal/Modeling|<font color="#ffffff">Modeling</font>]] | ||
| + | !style="text-align:center; background-color:#cd0000; border-width:0px; padding:3px;"|[[Team:Montreal/Notebook|<font color="#ffffff">Notebook</font>]] | ||
| + | |} | ||
| + | |||
| + | |||
| + | ==Protocol== | ||
| + | |||
| + | ===Gel Preparation=== | ||
| + | |||
1. Measure 50mL of TAE buffer and mix with 0.5g of Agarose powder. | 1. Measure 50mL of TAE buffer and mix with 0.5g of Agarose powder. | ||
2. Thoroughly mix the two in the flask and microwave the mixture for roughly about 1 minute (or until you see bubbling). | 2. Thoroughly mix the two in the flask and microwave the mixture for roughly about 1 minute (or until you see bubbling). | ||
| - | 3. Add 1.5µL of Ethidium Bromide and swirl until a fine mixture is seen. | + | 3. Add 1.5µL of Ethidium Bromide (light sensitive) and swirl until a fine mixture is seen. |
4. Pour the mixture onto the casting tray (make sure that each end is taped), with a comb inserted in one of the ends. | 4. Pour the mixture onto the casting tray (make sure that each end is taped), with a comb inserted in one of the ends. | ||
| - | 5. Wait for the gel to solidify over time. | + | 5. Wait for the gel to solidify over time. ''Optional: Loosely cover the gel with aluminum foil to reduce EtBr breakdown in light.'' |
6. Once hardened, remove the comb and load the gel into the electrophoresis box. | 6. Once hardened, remove the comb and load the gel into the electrophoresis box. | ||
| + | |||
| + | 7. Fill the reservoir with enough buffer to submerge the gel entirely. | ||
| + | |||
| + | ===Running the Gel=== | ||
| + | |||
| + | 1. To a DNA sample (15µL volume), add 2µL of loading dye. The loading dye acts to increase specific gravity and visibility of the DNA samples. | ||
| + | |||
| + | 2. Fill this entire volume (17µL) into the wells in the agarose gel using a P20 micropipette. Record the order of samples and their respective wells. | ||
| + | |||
| + | 3. Add between 2-5µL of DNA ladder to one empty well. It is sometimes preferable to add ladder to the two wells relatively far apart, to help line up the ladder with DNA bands. | ||
| + | |||
| + | 4. Apply a voltage of 100V across the gel for 45-60 minutes. Exceeding this voltage would excessively heat the gel and yield irregular DNA migration. ''Optional: Cover the reservoir with aluminum foil to reduce EtBr breakdown in light.'' | ||
| + | |||
| + | 5. When finished, remove the gel from the reservoir and photograph under UV light to confirm the presence and size of the DNA bands. | ||
Latest revision as of 19:57, 17 June 2008
| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook |
|---|
Protocol
Gel Preparation
1. Measure 50mL of TAE buffer and mix with 0.5g of Agarose powder.
2. Thoroughly mix the two in the flask and microwave the mixture for roughly about 1 minute (or until you see bubbling).
3. Add 1.5µL of Ethidium Bromide (light sensitive) and swirl until a fine mixture is seen.
4. Pour the mixture onto the casting tray (make sure that each end is taped), with a comb inserted in one of the ends.
5. Wait for the gel to solidify over time. Optional: Loosely cover the gel with aluminum foil to reduce EtBr breakdown in light.
6. Once hardened, remove the comb and load the gel into the electrophoresis box.
7. Fill the reservoir with enough buffer to submerge the gel entirely.
Running the Gel
1. To a DNA sample (15µL volume), add 2µL of loading dye. The loading dye acts to increase specific gravity and visibility of the DNA samples.
2. Fill this entire volume (17µL) into the wells in the agarose gel using a P20 micropipette. Record the order of samples and their respective wells.
3. Add between 2-5µL of DNA ladder to one empty well. It is sometimes preferable to add ladder to the two wells relatively far apart, to help line up the ladder with DNA bands.
4. Apply a voltage of 100V across the gel for 45-60 minutes. Exceeding this voltage would excessively heat the gel and yield irregular DNA migration. Optional: Cover the reservoir with aluminum foil to reduce EtBr breakdown in light.
5. When finished, remove the gel from the reservoir and photograph under UV light to confirm the presence and size of the DNA bands.
