Restriction enzyme digests
Materials required:
pP2X2-TOPO plasmid Buffer D (Promega)
pEGFP-N1 vector Megohm H2O
PstI Shrimp alkaline phosphatase
BamHI
Caution Heatblocks can reach temperatures of 95℃
Set up one of the following reactions using the appropriate plasmids and oligos 
2. Incubate for at 60 minutes at 37 ℃
3. Remove tube from heat block and pulse-spin.
4. Add 1 µl Shrimp alkaline phosphatase to backbone reaction only (if you add to insert, the ligation will fail).
5. Incubate for at 30 - 60 minutes at 37 ℃
6. Remove tube from heat block and pulse-spin.
7. Heat-inactivate the Shrimp alkaline phosphatase by incubation for 15 minutes at 65 ℃
8. Remove tube from heat block and pulse-spin.
Gel purification of DNA fragment on an agarose gel
Materials required
TAE running buffer
10mg/ml Ethidium bromide
DNA loading buffer
Megohm H2O
| Hot agarose, ethidium bromide and scalpel blades - Caution Ethidium bromide is a potent mutagen and suspected carcinogen. Molten agarose can cause nasty burns Ð handle with care. |
1. You will be provided with 100ml molten agarose at 60℃ (1.0 g agarose in 100 ml TAE).
2. Carefully add 10 µl Ethidium bromide (10 mg/ml stock).
3. Mix by swirling and pour the melted agarose (with comb in place) into the sealed gel former. Make sure the gel is on a flat surface. Try not to introduce air bubbles which will distort the running of the gel. Let the gel solidify for 30 min before loading.
4. Load samples into the wells
5. Run at 100 volts for 30 min to 2 h, or until the dye front nearly reaches the end of the gel.
6. Turn off the power supply and take a sterile scalpel blade, eppendorf and the gel to the UV transilluminator -you should be able to see the target fragment
