IGEM:MIT/2007/Notebook/2007-8-15

Agenda

1. Make electrocompetent BL-21 cells
2. Transform R0051.B0034 and plate
3. Transform I14032.B0034.CPX.B0014 into competent cells and plate
4. Transform F2620.B0034.CPX.B0014 into competent cells and plate
5. Run polystyrene-binding efficiency assay
6. Mercury removal assay

Making electrocompetent BL-21 Cells

Eh, not particularly fast compared to chemically competent cell if you cut the incubation on ice to 10 minutes.

Thank you, Barry, for letting us use your stash of BL-21s.

All the things transformed and/or plated today

All plasmids transformed into competent BL-21s

In 37C @ 1AM

Part                         Plasmid           Source                        Plate Antibiotic

F2620.B0034                  1AC3              8/14 ligation strip, #7       1 Cm; 1 Kan (control for insert re-ligation)

F2620.B0034                  3K3               8/14 ligation strip, #8       1 Cm (control for correctness of ligation experiment); 1 Kan
    note: (- control) no vector         

F2620.B0034.CPX              1AC3              glycerol stock                1 Cm

F2620.B0034.CPX#6-1.B0014    1AC3              miniprep, B-R 6-1             1 Cm     
    note: this plasmid is pick #1 of 6 minipreps and is derived from the colony PCR #6 line of F.B34.C plasmids.
          this pick was chosen because this plasmid had the clearest sequencing signals of the 6 minipreps.

F2620.B0034.CPX#8-5.B0014    1AC3              miniprep, B-R 6-5             1 Cm
    note: plasmid is pick 5 of 6, F.B34.C comes from cPCR #8.
          this pick was chosen because of its clear sequencing results.

I14023.B0034.CPS.B0014       1AC3              8/14 lig. strip, #5           1 Cm; 1 Kan (control for insert re-ligation)

I14032.B0034.CPX.B0014       1AC3              8/14 lig. strip, #2           1 Cm; 1 Kan (control for correctness of experiment)
    note: (- control) vector only, no inserts

I14032.B0034.CPX.B0014       3K3               8/14 lig. strip, #3           1 Cm (control for correctness of experiment); 1 Kan
    note: (- control) CPX.B0014 insert only, no vector or I.B34

I14032.B0034.CPX.B0014      2K3                8/14 lig. strip, #4           1 Cm (control for correctness of experiment); 1 Kan
    note: (- control) I14032.B0034 insert only, no vector or C.B14

Mercury Removal Assay Protocol

• Derived from Bae et al. Papers

1. Grow to OD of .3 in LB + Amp
2. Induce with 1mM IPTG
3. Induce for 2,3,4,5 hrs
4. Grow to OD of 1.0
5. Suspend in LB medium w/ 5uM Hg2+
6. Check [Hg2+] after 1 hr