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Purdue/2 September 2008
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| + | '''Restriction Digest of SOS and LacZ''' | ||
| + | |||
| + | 1. Mixed digestion solution: | ||
| + | *50uL DNA | ||
| + | *7uL NEB2 | ||
| + | *10.5uL ddH2O | ||
| + | *0.5uL BSA | ||
| + | *1uL of each enzyme | ||
| + | **SOS: EcoRI + SpeI | ||
| + | **LacZ: EcoRI + XbaI | ||
| + | **This makes SOS the small part cut out (192 bp) and put into the LacZ plasmid (~5000 bp...=LacZ + most of plasmid) | ||
| + | |||
| + | 2. Put mixtures at 37C for 1 hour, 25 min. | ||
| + | |||
| + | 3. Run through Agarose gel w/ DNA ladder, DNA loading dye (Bio-RAD) | ||
| + | *Mixed 7uL of loading dye w/ each 70uL tube | ||
| + | *10uL ladder in lane 1 | ||
| + | *Lanes 2-4: 20uL LacZ in each lane | ||
| + | *Lanes 5-7: SOS | ||
| + | *120V, 45 min. | ||
| + | |||
| + | 4. Overnight stain w/ Fast Blast | ||
| + | *5x | ||
| + | *RT | ||
| + | *dark | ||
| + | *rocking | ||
| + | |||
| + | |||
| + | '''Edited by Janie Stine''' | ||
Latest revision as of 17:05, 2 September 2008
Click Here to return to the notebook.
Restriction Digest of SOS and LacZ
1. Mixed digestion solution:
- 50uL DNA
- 7uL NEB2
- 10.5uL ddH2O
- 0.5uL BSA
- 1uL of each enzyme
- SOS: EcoRI + SpeI
- LacZ: EcoRI + XbaI
- This makes SOS the small part cut out (192 bp) and put into the LacZ plasmid (~5000 bp...=LacZ + most of plasmid)
2. Put mixtures at 37C for 1 hour, 25 min.
3. Run through Agarose gel w/ DNA ladder, DNA loading dye (Bio-RAD)
- Mixed 7uL of loading dye w/ each 70uL tube
- 10uL ladder in lane 1
- Lanes 2-4: 20uL LacZ in each lane
- Lanes 5-7: SOS
- 120V, 45 min.
4. Overnight stain w/ Fast Blast
- 5x
- RT
- dark
- rocking
Edited by Janie Stine
