IGEM:MIT/2007/Notebook/2007-8-15: Difference between revisions
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==Agenda== | ==Agenda== | ||
#Make electrocompetent BL-21 cells | #Make electrocompetent BL-21 cells | ||
#Transform R0051.B0034 and plate | #Transform R0051.B0034 and plate | ||
#Transform I14032.B0034.CPX.B0014 into competent cells and plate | #Transform I14032.B0034.CPX.B0014 into competent cells and plate | ||
#Transform F2620.B0034.CPX.B0014 into competent cells and plate | |||
#Run polystyrene-binding efficiency assay | #Run polystyrene-binding efficiency assay | ||
#Mercury removal assay | #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. | |||
==Made a 5mL LC of BL-21 so we can make our own competent cells and not bother Barry anymore== | |||
==All the things transformed and/or plated today== | |||
All plasmids transformed into competent BL-21s | |||
<pre> | |||
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 | |||
</pre> | |||
==Mercury Removal Assay Protocol== | ==Mercury Removal Assay Protocol== |
Revision as of 22:23, 15 August 2007
Agenda
- Make electrocompetent BL-21 cells
- Transform R0051.B0034 and plate
- Transform I14032.B0034.CPX.B0014 into competent cells and plate
- Transform F2620.B0034.CPX.B0014 into competent cells and plate
- Run polystyrene-binding efficiency assay
- 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.
Made a 5mL LC of BL-21 so we can make our own competent cells and not bother Barry anymore
All the things transformed and/or plated today
All plasmids transformed into competent BL-21s
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
- Grow to OD of .3 in LB + Amp
- Induce with 1mM IPTG
- Induce for 2,3,4,5 hrs
- Grow to OD of 1.0
- Suspend in LB medium w/ 5uM Hg2+
- Check [Hg2+] after 1 hr