From 2008.igem.org
June 2008
June 2
- Growth of I-brick on culture
- Midi-prep of both I and J brick followed by gel
- Gel indicates no presence of DNA, will be confirmed by spectrophotometric assay
June 3
- Seeding of 5mL cultures of both I and J brick
- Identity of colonies on I brick plates is suspect, must ensure that eventual DNA gel confirms exact restriction digest
June 4
Midiprep was done on the I and the J-brick. Once the isopropanol was added, the J-brick midiprep looked clear (no DNA was eluted). DNA gel needs to be done to confirm presence of DNA in both cases.
June 9
- Seeded J and I-brick re-seeded for Maxi-Prep
- Gel failed to confirm presence of previously collected DNA samples of J and I-brick, will be repeated following Maxi-Prep.
June 10
Since no growth was observed in the I-brick culture, the I brick was re-seeded.
The J-brick was diluted in 500-ml of LB broth(for a Maxiprep to be done the next day).
June 11
- Maxiprep of the J-brick.
- Restriction digest of J-brick.
- Dilution of I-brick in 500-ml of LB broth.
June 12
- Maxiprep of the I-brick.
- Restriction digest and gel of June 11th and June 12th I-brick and J-brick DNA using EcoR1. Bands revealed at roughly 4000bp and 2500bp for I-brick (expected 2652bp and 3939bp). J brick single band that was not informative, new digestion to be completed tomorrow.
June 16
- I-brick was seeded and diluted over last two days, but there was insufficient growth so it will be left to grow one more day before performing another midi-prep. This is to compliment the already successful Maxi-Prep that gave low concentrations of DNA.
- Another gel was performed of previous J-brick preps that confirmed the absence of the desired plasmid, no DNA was detected when digested with EcoR1.
- J-brick was re-transformed into TOP10 chemically competent cells and then plated on Amp+ plates.
June 17
- The J-Brick was re-seeded since when a gel was ran on the J-Brick, no DNA was observed.
- J-Brick plate was left out to maximize its growth overnight.
- More antibiotic was added to the I-Brick culture (since it did not grow in LB broth for the past two days) and it was transferred from a Falcon tube to an Erlenmeyer Flask to maximize its growth.
June 18
- Midiprep of I-brick (1 sample)
- Miniprep of J-brick (2 samples)
- Restriction digest and gel electrophoresis of both J-brick miniprep samples (with SpeI/XmnI+BSA and EcoRI) and the I-brick midiprep sample (with XbaI+BSA); total of five wells + 1 kb ladder. Only ladder showed under UV light.
June 19
- Re-seeded J-brick from 2008-06-16 plates for miniprep (two vials, JA and JB)
- Re-seeded I-brick for miniprep (IA and IB)
- Included two controls - LB and LB/amp+
June 20
- No significant growth in 2008-06-19 control vials or IA,
- Regular growth in vials IB, JA
- Different E.coli morphology in JB; under same environment, bacteria in this vial aggregated into one group at the bottom of the vial
- Redo miniprep of J-brick (JA) and I-brick (IB
"
