Team:UNIPV-Pavia/Methods Materials/Electrophoresis
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*'''Ethidium bromide''' | *'''Ethidium bromide''' | ||
*'''Loading buffer 10x Blue Juice, Invitrogen''' | *'''Loading buffer 10x Blue Juice, Invitrogen''' | ||
- | *'''TBE (Tris/Borate/EDTA buffer) | + | *'''TBE (Tris/Borate/EDTA buffer) 5x (final volume 1 liter)''' |
- | + | ||
**'''54 gr Tris''' | **'''54 gr Tris''' | ||
**'''27.5 gr Borate''' | **'''27.5 gr Borate''' | ||
Line 51: | Line 50: | ||
*'''Transilluminator''' | *'''Transilluminator''' | ||
<br> | <br> | ||
- | *Prepare | + | *Prepare agarose gel in 1x TBE buffer |
*Add ethidium bromide (using gloves and face mask for your safety): | *Add ethidium bromide (using gloves and face mask for your safety): | ||
**1 µl in the small size agarose gel (70 ml) | **1 µl in the small size agarose gel (70 ml) | ||
**2 µl in the middle size agarose gel (150 ml) | **2 µl in the middle size agarose gel (150 ml) | ||
- | **4 µl in the big size agarose gel ( | + | **4 µl in the big size agarose gel (250 ml) |
*Cast the gel, insert the well-forming comb and let it polymerize | *Cast the gel, insert the well-forming comb and let it polymerize | ||
*Add the loading buffer to each sample | *Add the loading buffer to each sample | ||
- | *Load the samples and 8 µl marker | + | *Load the samples and 8 µl of marker |
*Set to 70-100 volts and electrophorese for the required amount of time | *Set to 70-100 volts and electrophorese for the required amount of time | ||
*Use UV-light to look at the bands | *Use UV-light to look at the bands | ||
- | *Take a picture of the gel, if needed (not when bands have to be cut) | + | *Take a picture of the gel, if needed (not when bands have to be cut!!!) |
<br> | <br> |
Revision as of 08:27, 24 June 2009
Electrophoresis
(estimated time: 2 hours)
Materials needed:
- DNA samples
- Ethidium bromide
- Loading buffer 10x Blue Juice, Invitrogen
- TBE (Tris/Borate/EDTA buffer) 5x (final volume 1 liter)
- 54 gr Tris
- 27.5 gr Borate
- 20 ml EDTA 0.5 M (pH 8)
- Marker (1 kb DNA Ladder, Promega)
- Face mask and gloves
- Electrophoresis apparatus
- Transilluminator
- Prepare agarose gel in 1x TBE buffer
- Add ethidium bromide (using gloves and face mask for your safety):
- 1 µl in the small size agarose gel (70 ml)
- 2 µl in the middle size agarose gel (150 ml)
- 4 µl in the big size agarose gel (250 ml)
- Cast the gel, insert the well-forming comb and let it polymerize
- Add the loading buffer to each sample
- Load the samples and 8 µl of marker
- Set to 70-100 volts and electrophorese for the required amount of time
- Use UV-light to look at the bands
- Take a picture of the gel, if needed (not when bands have to be cut!!!)