Registry/Measurement kit/Notebook/2007-6-14

Registry/Measurement_kit/Notebook

To Do

1. Update Registry/Measurement kit/Glycerol box
2. Miniprep 5 constructs
3. Digest 5 constructs
4. 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

Knight:Restriction_Digest

• Will do tomorrow

• Measured concentration (ng/µL) of DNA with Nanodrop:

1. E0240 - 54.2
2. R0040 - 27.1
3. I13401 - 113.1
4. J04650 - 213.5
5. B0032 - 45.2

• How to digest/combine constructs?
  • 3-way or 2-way (suffixing) ligation?
    • Will try 2-way 3-way. Outline of steps:

1. Digest
2. "PCR cleanup"
3. Ligate

Digest plan

1. E0240 done.
2. R0040 at EcoRI, SpeI.
3. I13401 at XbaI, PstI.
4. J04650 at XbaI, PstI.
5. B0032 at EcoRI, SpeI.

• Jason R. Kelly: This looks like a plan for 3 way ligations (which is fine, just not what it says above), do we have the backbone vector that you want to put these into? What enzymes does it need to be cut with? Do we have plates for selection of the backbone?

• Nishant M. Bhat: You're right, my mistake. It has been corrected.