IGEM:MIT/2007/Notebook/2007-7-17

=Agenda=
 * Check sequencing results of second (uncontaminated) set of miniprepped 3K3+F2620+B0034
 * Digest CPX, 3K3+F2620+B0034 (if sequencing is correct), and a hi-copy plasmid for 3A assembly
 * Ligate digested CPX, 3K3+F2620+B0034, and hi-copy plasmid
 * Test Standard Assembly of pSB3k3 with F2620, B0034, E1010 using AHL
 * LC grown overnight

Digestion of CPX for 3A
5 µl      CPX (diluted to 1µg/5µl) 0.5 µl    BSA 5 µl      NEB3 Buffer 1 µl      XbaI 1 µl      PstI 37.5 µl   H2O -- 50 µl     Total

Digestion of (F2620 and B0034) for 3A
37 µl     (F2620 and B0034) @ 27 ng/µl 0.5 µl    BSA 5 µl      NEB2 Buffer 1 µl      EcoRI 1 µl      SpeI 5.5 µl    H2O -- 50 µl     Total


 * Digested pSB1AC3: 15 µl @ 125 ng/µl
 * Digestions placed in 37C @ 11am

Changes to the OWW "Transforming Chemically Competent Cells" Protocol for TK Cells

 * All steps are the same, except for the following:
 * In step 2, Note -- TK said that less than 5% of the solution should be cell volume
 * Step 4 -- change to 45 to 50 seconds at 42 degrees
 * Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or Cm resistant cells

Miniprep Results for 5mL LCs of pT040 made on 7/16
Colony  DNA Conc. 1       60.6 ng/µL 2       64.3 ng/µL 3       78.2 ng/µL 4       57.9 ng/µL

eppendorfs containing the purified plasmids are labeled:

iGEM 7/17 pur. pT040 LC # Concentration

Miniprep of PCR Purified Double Digested CPX and F2620+B0034
CPX (X, P): 7.6 ng/µl F2620+B0034 (E, S): 12.0 ng/µl

Ligation of F2620+B0034 and CPX into pSB1AC3
Ligation Mix: 1 µl     53ng backbone (pSB1AC3) 6 µl     72ng F+B 6.2 µl   47ng CPX 0.5 µl   T4 ligase 2.0 µl   10x T4 ligase buffer 4.3 µl   H20

20 µl    Total

Negative Control(Backbone only, no inserts): 1 µl     53ng backbone (pSB1AC3) 0.5 µl   T4 ligase 2.0 µl   10x T4 ligase buffer 16.5 µl  H20

20 µl    Total

Put into 16C at 4PM into Thermal Gradient Cycler

=Transformations=

CPX Transformation

 * Transformed 1 ul into TK cells

GFP Tranformation

 * Transformed 1 ul into TK cells

FhuA Ligated Insert Transformation

 * 10 ul each of PHIE6 and PICCS8 into TK cells

=AHL RFP Testing= Failed Possible Reasons:

=Mer Construct Digestion=
 * 2 ul EcoR1 Buffer
 * 2 10X BSA
 * .5 EcoR!
 * .5 Spe1
 * 9.6 ul DNA
 * 5.4 H20