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