IGEM:MIT/2007/Notebook/2007-7-11

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Contents

Agenda

1.) Miniprep Backbone LC, DD eRFP (+, - controls), DD miniprep backbone (do we need to gel or pcr purify?), Standard assembly by running gel, purifying gel, ligation, transformation
2.) Make LC of MerPR and MerR Plates
3.) Send final transformation miniprep for sequencing

Diluted LC's of Transformed XL-1

  • Used Samples #3 and #4 from overnight LC's
  • Overnight LC's are colonies picked from source plate: 7/9 XL1-blue transformed with pSB3K3+F2620+B0034, 20µL of culture plated on LB+Kan
  • 1:5 dilution: therefore, we now have 25µl for each cell line
    • JH: Accidentally diluted line #3 6X (30µL total). Line #4 is still 5X dilution.
  • Placed in 37C around 10am

Notes on Future Use of Transformed XL-1

  • Miniprep
  • Some for sequencing
  • standard assembly with eRFP
  • Save some for standard assembly with sticky insert
  • Run a diagnostic digest (use EcoRI and PstI to digest, check length by running gel)
  • Permanent glycerol stock (2ml for 2 stocks)

DD part E1010

  • Digested with EcoRI and SpeI
  • Used 121.5 ng/µL eppendorf of E1010
  • Mixture:
    • 8.23µL of DNA
    • 5µL of EcoRI Buffer
    • 2µL of EcoRI
    • 2µL of SpeI
    • 0.5µL of BSA
    • 32.27µL of H2O
  • 3 Hrs in 37C room. Put in at 11:50AM
  • Redid digestion later, needed to use XBaI and PstI rest. enzymes

Made More 1% Agarose in TAE Buffer

  • 1L flask contains: 1L of TAE Buffer and 10 mg of agarose powder each
  • TAE buffer can be found above the sink
  • Agarose powder can be found on chemical powder shelf rm 558e. Containers are alphabetized.
  • Flask kept in the 55C box next to Jason's bay.

Results for Transformed XL-1 (pSB3K3 + F2620 + B0034) Miniprep

  • Used dilutions of LC #4 (made earlier today)
Culture    Concentration
1          98.9 ng/µL
2          144.6 ng/µL
3          139.6 ng/µL
4          133.4 ng/µL
5          132.6 ng/µL

Diagnostic Double Digestion of Today's Miniprep of pSB3k3+F2620+B0034

3.6 µl DNA Miniprep #3 (139.6 ng/µl)
0.2 µl BSA
2.0 µl NEB3 Buffer
0.5 µl EcoRI
0.5 µl PstI
13.2 µl H2O

20µl Total
  • Placed in 37C for 3 hrs starting 6:30pm

Double Digestion for Standard Assembly with eRFP

7.20 µl DNA Miniprep #3 (139.6 ng/µl)
0.5 µl BSA
5.0 µl NEB2 Buffer
1.0 µl SpeI
1.0 µl PstI
35.3 µl H2O

50µl Total
  • Made 2 digestions, both with same contents
  • Placed in 37C for 3 hrs starting at 6:30pm
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