Registry/Measurement kit/Notebook/2007-6-14: Difference between revisions
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*How to digest/combine constructs? | *How to digest/combine constructs? | ||
**3-way or 2-way (suffixing) ligation? | **3-way or 2-way (suffixing) ligation? | ||
***Will try 2-way. | ***Will try 2-way. Outline of steps: | ||
# Digest | |||
# "PCR cleanup" | |||
# Ligate | |||
===Digest plan=== | |||
# E0240 done. | |||
# R0040 at EcoRI, SpeI. | |||
# I13401 at XbaI, PstI. | |||
# J04650 at XbaI, PstI. | |||
# B0032 at EcoRI, SpeI. | |||
*Remember to discuss group recommendations. |
Revision as of 13:36, 14 June 2007
To Do
- Update Registry/Measurement kit/Glycerol box
- Miniprep 5 constructs
- Digest 5 constructs
- re-Evaluate design based on recommendations from group meeting
Mini-prep of cultures
- made glycerol stocks - 1mL of culture in each --> -80 freezer.
- placed remaining culture in 1.5mL tubes, spun at 5k for 4 min
Notes
- extra reagents in Kit Reagents. 3rd shelf in 2nd room fridge
- protocol in blue booklet
- get ~150mL beaker for supernatant
- be sure to shake the reagents from the kit a little before using them
- resuspend the whole culture (not individual pellets) in 250µL of P1.
Digest of constructs
- will do tomorrow
- Measured concentration (ng/µL) of DNA with Nanodrop:
- E0240 - 54.2
- R0040 - 27.1
- I13401 - 113.1
- J04650 - 213.5
- B0032 - 45.2
- How to digest/combine constructs?
- 3-way or 2-way (suffixing) ligation?
- Will try 2-way. Outline of steps:
- 3-way or 2-way (suffixing) ligation?
- Digest
- "PCR cleanup"
- Ligate
Digest plan
- E0240 done.
- R0040 at EcoRI, SpeI.
- I13401 at XbaI, PstI.
- J04650 at XbaI, PstI.
- B0032 at EcoRI, SpeI.
- Remember to discuss group recommendations.