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

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
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
 * Used dilutions of LC #4 (made earlier today)

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

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 * 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

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 * 50µl Total


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