Team:Montreal/Notebook/June

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, J_A and J_B)
• Re-seeded I-brick for miniprep (I_A and I_B)
• Included two controls - LB and LB/amp+

June 20

• No significant growth in 2008-06-19 control vials or I_A,
• Regular growth in vials I_B, J_A
• Different E.coli morphology in J_B; under same environment, bacteria in this vial aggregated into one group at the bottom of the vial
• Redo miniprep of J-brick (J_A) and I-brick (I_B); eluted 100µL of DNA for each

June 25

• Seeding of Cultures of I and J Brick (2 each) with 2 controls (1 Amp+, 1 Amp+/Kan+)
• Dilution of J culture into 100mL with LB for midi-prep
• No growth of I-seeding, re-seeded
• No growth in controls

June 26

• Midi-prep of J brick culture
• No growth of second I-seeding, will be re-transformed later

June 30

• J-brick re-seeded, previous midi-prep failed
• I-brick also seeded, no growth after 48 hours and previous 5mL culture tubes from weekend (72 hours) show no growth as well. Does not bode well...