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

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

Digestion of (F2620 and B0034) for 3A

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

• Digested pSB1AC3: 15µl @ 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 Cu 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