IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-20

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Miniprep of J04500

Played Jazz in the lab & miniprepped .2 mL J04500 as per the todo list left yesterday.

Notes: Eluted in H20

Left 4 miniprep tubes in our fridge. nanodrop results as follows: 1: 12.6 2: 13.2 3: 15.6 4: 14.2

The results are just about what we get every time.

Digests will follow later after dinner.

Anyone want to take a crack at the presentation for tomorrow while I do the digests? Or anyone want to do the digests and I'll take care of the presentation? Let me know.

Digestion of J04500 with X-P & S-P, KaiA/B/C with X-P

Performed the digests of the midiprepped KaiA/B/C and the miniprepped J04500 from earlier today. Ran two digests of each of the following: J04500 with X-P J04500 with S-P KaiA/B/C with X-P

J04500 Digest Protocol

Reaction X-P:

  • 21.25 µL DNA
  • 2.5 µL NEBuffer 3
  • 0.5 µL XbaI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA

Reaction S-P:

  • 21.25 µL DNA
  • 2.5 µL NEBuffer 2
  • 0.5 µL SpeI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA

Master X-P:

  • 10 µL NEBuffer 3
  • 2 µL XbaI
  • 2 µL PstI
  • 1 µL 100x BSA

Master S-P:

  • 10 µL NEBuffer 2
  • 2 µL SpeI
  • 2 µL PstI
  • 1 µL 100x BSA

Pipette 3.75 µL into each reaction. Digested for 16 hours at 37C in PCR-?.

KaiA/B/C X-P Digest Protocol

Reactions X-P:

  • 8 µL midiprepped DNA
  • 2.5 µL NEBuffer 3
  • 0.5 µL XbaI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA
  • 13.25 µL H2O

Master X-P:

  • 17.5 µL NEBuffer 3
  • 3.5 µL XbaI
  • 3.5 µL PstI
  • 1.75 µL 100x BSA
  • 92.75 µL H2O

Pipette 17 µL into each reaction. Digested for 16 hours at 37C in PCR-?.

Diagnostic digest of 13 minipreps from 2006-8-17 ligation

We performed \X-P digests of the 13 minipreps as per yesterday's to-do list.

Reaction:

  • 21.25 µL DNA
  • 2.5 µL NEBuffer 3
  • 0.5 µL XbaI
  • 0.5 µL PstI
  • 0.25 µL 100x BSA

Master:

  • 37.5 µL NEBuffer 3
  • 7.5 µL XbaI
  • 7.5 µL PstI
  • 3.75 µL 100x BSA

Pipette 3.75 µL into each reaction.

Digested for 8 hours at 37C in PCR-1.