Haynes Lab:Notebook/Jan/2015/03/27

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03/27/15

  • Cultures - BD006/MV2 colonies (from 3/26/15 ligations), 5 mL each (4 total)
  • Plasmids - DPRE minipreps (5 mL cultures)

Plasmids - DPRE minipreps

  • ---Karmella 15:12, 27 March 2015 (EDT):
  • Sigma GenElute Miniprep kit.
  • Elute with 70μL elution buffer
  1. DPRE a
  2. DPRE b
  • Digests (Fermentas FD)
    • E/S to prep for insertion upstream of BD004
    • Also digest BD004 w/ E/X
Reagent Volume Expected:
1,2. DPRE (E/S) = ###, 3200
3. BD004 (E/X) = 7318 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Conclusions
    • MV2 is correct
    • BD004 - most likely missing the D11.12 fragment. Sequence results from Meli'sa's past work agrees with the short fragment size on the gel.
    • BD006 is correct
  • Miniprep conc.'s
    • Combined the pairs of preps. Pairs look identical.
Sample OD260 260/280 ng/μL
1. MV2 mp 0.132 1.85 132.262
2. BD004 mp 0.144 1.91 143.967
3. BD006 mp 0.078 1.83 78.413



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