Purdue/2 September 2008
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(Difference between revisions)
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**This makes SOS the small part cut out (192 bp) and put into the LacZ plasmid (~5000 bp...=LacZ + most of plasmid) | **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 | + | 2. Put mixtures at 37C for 1 hour, 25 min. |
- | 3. | + | 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 | + | 4. Overnight stain w/ Fast Blast |
- | + | ||
- | + | ||
'''Edited by Janie Stine''' | '''Edited by Janie Stine''' |
Revision as of 17:05, 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
Edited by Janie Stine