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Team:Brown/Notebook/July17
From 2010.igem.org
Saturday, July 17 2010
Checked transformed plates from yesterday:
- RFP control, 100 picograms DNA
- Null plate – lawn
- Amp+ plate – 8 colonies + some sort of fungus/fuzzy contamination.
So, we are redoing it one more time, in six different conditions:
- 100 picograms DNA
- 1 ng DNA
- 10 ng DNA
- DNA = one control RFP, one empty pNoTat (at 83 ng/µl)
- 1/10 dilution of control RFP to get 1 ng/µl DNA.
Incubate at 37°C overnight again.
6:00 PM: removed overnight of BBa_K191003 to create a glycerol stock of it, and to miniprep it to isolate DNA.
Resuspended 1 mL of overgrown overnight into 4 mL of fresh LB and 3.57µl kanamycin to make 5 mL culture with Kanamycin at 25 µg/mL and began incubating.
Spin down 5 mL of overnight, overgrown culture at 9.3 x 103 RPM for 4.5 minu, and then followed Qiagen miniprep protocol with 5 aliquots.
Nandropped to find DNA concentration: 105.6 ng/µl.
7:20 PM: retrieved and began thawing in ice bucket: 7 aliquots of XL1Bs from yesterday and 5 aliquots of old “competent” Bl21s but realized that we are ignorant of the vectors containing the pats to be transformed, so returned DNA to freezer and dumped aliquots.
7:45 PM: Aliquoted log-phase cells into four microcentrifuge tubes, pelleted the cells, discarded old LB, resuspended in 900 µl new LB, added to a cryogenic tube with 900 µl 40% glycerol stock and placed in -80°C.
