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