IGEM:MIT/2007/Notebook/2007-7-18: Difference between revisions
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*Poured 29 LB+Cm plates | *Poured 29 LB+Cm plates | ||
*Cm plates have green labels | *Cm plates have green labels | ||
==Meeting Notes== | |||
*Run a sensitivity easy experiment | |||
*HgCl2 MSDS | |||
**Ask tom for HgCl2, or even chemical room | |||
*Reshman has the DNA for Leucine zipper | |||
**CPX to display ? | |||
*''''Transcriptional Terminator'''' | |||
*B0010 and B0012 | |||
**B0015 is the double terminator and works very well. | |||
*Get T7 antibody, order | |||
**Think of getting fluorescent version of the body, secondary version that is fluorescent | |||
**Endy lab or Brian's lab. Has origin antibody. | |||
*Could use FLAG tag in FhuA, or HA tag, needs to work internally; CMiC | |||
*Get EC20 display, and CCG? | |||
**LPP-OMP-A | |||
First Lorenzo, fused a single chain antibody. Antibody, can get complement; | |||
*Make Cell maps | |||
Input then output. | |||
Cysteines: will not export proteins that have internal cysteine bonds. Periplasm is where they farm. No export through that system. | |||
Washing: | |||
Negative control | |||
EBS, salt conditions | |||
Absorption on Spec | |||
1 OD of cells | |||
Take 2 mL |
Revision as of 11:53, 18 July 2007
Agenda
- Transform tk's competent cells with yesterday's 3A assembly results (F2620+B0034 and CPX insert into vector pSB1AC3)
- Make LB+Cm and LB+Cm+Kan plates
- Liquid cultures of eight tubes made and put in warm room -- for making glycerol stocks
- 2 tubes of DB3.1 (Kan+)
- 2 tubes of transformed E1010 from resistance test plate (Kan+)
- 2 tubes of transformed F2620 from resistance test plate (Amp+)
- 2 tubes of transformed B0034 from resistance test plate (Amp+)
- All should be taken out tomorrow morning and used to make glycerol stocks
- Meeting with TK and Drew
Transformed tk's competent cells
- Aliquoted one of the original vials into eight eppendorfs, 100µL each.
- Did 3 sets of transformations:
- 2 of yesterday's 3A assembly product (plasmid contains: F2620+B0034+CPX in pSB1AC3 backbone)
- 1 negative control (pSB1AC3 digested with EcoRI and PstI)
- Followed our Transformation protocol (tk's modified protocol)
- Transformations were put into 37C room at 11:30 AM and incubated for 2 hrs
Poured Plates, again
- Used stringent working concentrations:
- 10µg of Kanamycin per mL of LB
- 25µg of Chloramphenicol per mL of LB
- Poured 5 LB+Cm+Kan plates
- Poured 29 LB+Cm plates
- Cm plates have green labels
Meeting Notes
- Run a sensitivity easy experiment
- HgCl2 MSDS
- Ask tom for HgCl2, or even chemical room
- Reshman has the DNA for Leucine zipper
- CPX to display ?
- 'Transcriptional Terminator'
- B0010 and B0012
- B0015 is the double terminator and works very well.
- Get T7 antibody, order
- Think of getting fluorescent version of the body, secondary version that is fluorescent
- Endy lab or Brian's lab. Has origin antibody.
- Could use FLAG tag in FhuA, or HA tag, needs to work internally; CMiC
- Get EC20 display, and CCG?
- LPP-OMP-A
First Lorenzo, fused a single chain antibody. Antibody, can get complement;
- Make Cell maps
Input then output.
Cysteines: will not export proteins that have internal cysteine bonds. Periplasm is where they farm. No export through that system.
Washing:
Negative control
EBS, salt conditions
Absorption on Spec
1 OD of cells
Take 2 mL