Purdue/2 September 2008
From 2008.igem.org
(Difference between revisions)
(New page: Click Here to return to the notebook.) |
|||
(3 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
[[Team:Purdue/Notebook | Click Here to return to the notebook.]] | [[Team:Purdue/Notebook | 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''' |
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