Team:BU Wellesley Software/Notebook/ShannonNotebook

WEEK 1: 6/06-6/10/2011

• Included a boot camp.
• Different topics were discussed (i.e. biobricks, gene regulation, differences between eukaryotes and prokaryotes).
• Boot camp also included a computer science component (focused on Clotho).
• Miniprepes were done on previously transformed BFP2. After minipreps, the Nanodrop was used to quantify the DNA. Minipreps did not have high DNA concentration, but were run on a gel (to show presence of DNA). Most lanes appeared empty (bands of DNA were not visible).
• Transformations of Pbad were conducted.
  • The following day, transformations appeared to be contaminated. They looked significantly different than usual, but were still were used to produce overnight cultures.
  • To ensure that E.coli was present gram staining was performed. Staining showed that no E.coli was present in the cultures. Nanodrop was used to quantify the DNA from the overnight cultures.
    • The results from the Miniprep/Nanodrop varied (some overnight cultures had a decent DNA concentration, others didn’t). Even though yields were not great the samples were run on a gel. However no bands were visible.

WEEK 2: 6/13-6/17/2011

• Overnight cultures were made from the previous week’s transformations.
  • cultures didn’t grow properly (only UV plasmid).
• An issue with the plates was discovered. All previous cultures were done on Ampicillian plates, and most were not growing well. We switched to a different antibiotic (Kanamycin) and all proceeding cultures grew well.
• New Ampicillian plates were made.
• A side project involved ligating GFP (Bba_J52625) and terminator (Bba_B0015) together for future use.
• Transformations were completed (involved transforming several different biobrick parts).
  • Two seemed to look pinkish, especially towards the center of each colony.
  • Successful transformations were then used to produce more overnight cultures.

WEEK 3: 6/20-6/24/2011

• Overnight cultures were made for the following: Bba_B0015.1, Bba_B0015.2, Bba_B0015.3, Bba_B0015.4, Bba_R0040.1, Bba_R0040.2, Bba_J52028.1, Bba_J52028.2, Bba_J52028.3,and Bba_J52028.4.
• Nanodrop was also conducted. Chart below shows values from 6/23/2011.

• Ligation was also performed. I tried ligating BFP (Bba_K156010) with terminator (Bba_B0015.2.2). Transformation was done of this ligation later that day. The following morning the transformation plates did not have any colonies on them.
• Minipreps were conducted on 6/23/2011’s overnight cultures and Nanodrop quantification was then performed. Results are shown below in the following chart:

WEEK 4: 6/27-7/01/2011

• Overnight cultures made using the following parts: Bba_J23100, Bba_J23101, Bba_E0240, Bba_E0430, Bba_R2000, Bba_I13453, Bba_R4000, and Bba_I14033.
• Restriction digest was done on the following: Bba_R2000.1, Bba_R2000.2, Bba_R0040.1, Bba_E0240.1, Bba_E0240.2, and Bba_J52028.3.
• transformations were done using the following ligations:
  • Bba_E0240.1 + Bba_R2000.1
  • Bba_E0240.1 + Bba_R2000.2
  • Bba_E0240.2 + Bba_R2000.1
  • Bba_R0240.2 + Bba_R2000.2
  • Bba_E0240.1 + Bba_R0040.1
  • Bba_E0240.2 + Bba_R0040.1
• Minipreps and Nanodrop were done on the previous day’s overnight cultures. Results are shown below.

• Minipreps and Nanodrop were done on the overnight cultures that were done the previous day. Results of Nanodrop are shown in the following chart.

• The 2.5A, B, and C all refer to various samples that were ligated with 2.5 uL of ligase. For example, sample 1A was ligated with 1 uL of ligase.
• Glycerol stocks were also made from the week's successful overnight cultures.
• Plasmid preps were done on that week’s successful transformations.
• To increase DNA concentration in ligations, I tried ethanol precipitation with GFPc.

WEEK 5: 7/04-7/08/2011

• Transformations were done of various parts in the beginning of the week (including BFP + Term and YFPc + R2000).
• Ligation was also performed on ECFP + Term and EYFP + Term. Transformations were then done using those ligations.
• However, transformations were not successful (no colonies grew).
• Plasmid preps were done earlier in the week. Nanodrop was then done. Yielded decent values (ranging from 25.6-61.9 ng/uL).

WEEK 6: 7/11-7/15/2011

• Restriction digest was done on ECFP and EYFP (both cut with E + S).
• Gel was run later that day of RD products. However, no bands were visible.
• Re-did restriction digest of ECFP and EYFP. Ran a gel and did gel extraction. ECFP had a concentration on 41.8 ng/uL and EYFP had a concentration of 18.8 ng/uL.
• To ensure that ligations were not producing false positives, CIP was used on the backbone.
• Ligations were done using a CIP backbone and a non-CIP backbone.
• Transformations were then done using both CIP and non-CIP backbone and sat on the bench top over the weekend.

WEEK 7: 7/18-7/22/2011

• Transformations from the previous week worked (both CIP and non-CIP).
• Made overnight cultures using the CIP and non-CIP transformations.
• Decided to troubleshoot ligations by trying different ratios and different lengths of time.
• Transformed ligations that were used to troubleshoot.
• Overnight cultures did not grow. Streaked plates from glycerol stocks and made overnight cultures.
• Attempted to ligate ECFP + Term and EYFP + Term. Also transformed the ligations. Put in the 37 C overnight.