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.

WEEK 8: 7/25-7/29/2011

• Transformations from the previous week seemed to work. Did plasmid preps. Some samples looked clear (some did not appear to have much growth). Yielded poor DNA concentrations (ranging from 2.0 t0 19.4).
• Re-ligated ECFP + Term and EYFP + Term. Transformed the samples and put in the 37 C overnight. Ultimately, these transformations did not grow.
• Restriction digest was done on EYFP, ECFP, and Terminator. Only band that cut was Terminator. Did gel extraction and Nanodrop.
• Overnight cultues were made from streaked plates. Minipreps were done and results were decent (ranging from 26.7 ng/uL to 42.1 ng/uL).

WEEK 9: 8/01-8/05/2011

• Restriction digest was done on ECFP, EYFP, and Terminator. Stored in the -20 C overnight. Overnight cultures were made for ECFP and EYFP.
• Minipreps were done for the overnight cultures that were done the previous day. Nanodrop was then done (produced decent values, ranging from 28.3 ng/uL to 33.0 ng/uL
• Ran a gel from 8/01/2011’s restriction digest. Was done in a 20 well comb.
  • • Lane 10: Cut EYFP
    • Lane 11: Cut EYFP
    • Lane 12: Uncut EYFP
    • Lane 13: Cut Terminator
    • Lane 14: Cut Terminator
    • Lane 15: Uncut Terminator

• EYFP appeared to cut. Cut out two lower bands and did gel extraction.
• Restriction digest of ECFP and EYFP was performed. This time, I tried using a higher concentration of DNA in the restriction digest. Instead of the usual 500 ng, I tried doubling the concentration to 1000 ng (one sample had 700 ng instead of 1000 ng). Previously, all samples were cut with E + S to ligate with terminator. However, I had not had any success, so I cut with X + P to ligate with RBS instead. This digest was also done overnight.
• Transformations were also done with ECFP (E0020) and EYFP (E0030) from the iGEM plates. Plated 100 uL of dilute cells and then 100 uL of concentrated cells. The next day the plates looked good. Put them in the 4 C.

WEEK 10: 8/08-8/12/2011

• Overnight cultures were made for 8/05/2011’s transformations.
• Gel was run from the restriction digest that was done in the later part of the previous week. All samples cut. Did gel extraction.
  • • Lineup for gel.
  • Lane 1: Ladder
  • Lane 2: Cut E0020.2
  • Lane 3: Cut E0020
  • Lane 4: Uncut E0020
  • Lane 5: Cut E0032.2
  • Lane 6: Uncut E0032.2
  • Lane 7: Cut E0032
  • Lane 8: Uncut
  • Lane 9: Vanessa
  • Lane 10: Vanessa
  • Lane 11: Vanessa
  • Lane 12: empty

• Minipreps were done for overnight cultures that were prepared the day before. Nanodrop results are shown below.

• Did restriction digest on all samples (ECFP A1 – EYFP B2). Did two restriction digest samples per one miniprep sample (one sample cut with second cut with X + P).
• Ran a gel with restriction digest samples.
• Lineup for wells:
  • 12 well.
    • Lane 1: Ladder
    • Lane 2: Cut ECFP A1, E + S
    • Lane 3: Cut ECFP A1, X + P
    • Lane 4: Uncut ECFP A1
    • Lane 5: Cut ECFP A2, E + S
    • Lane 6: Cut ECFP A2, X + P
    • Lane 7: Uncut ECFP A2
    • Lane 8: Cut ECFP B1, E + S
    • Lane 9: Cut ECFP B1, X + P
    • Lane 10: Uncut ECFP B1
    • Lane 11: Cut ECFP B2, E + S
    • Lane 12: Cut ECFP B2, X + P

• • 20 well.
    • Lane 1: Ladder
    • Lane 2: Uncut ECFP B2
    • Lane 3: Cut EYFP A1, E + S
    • Lane 4: Cut EYFP A1, X + P

•** Lane 5: Uncut EYFP A1

• • • Lane 6: Cut EYFP A2, E + S
    • Lane 7: Cut EYFP A2, X + P
    • Lane 8: Uncut EYFP A2
    • Lane 9: Cut EYFP B1, E + S
    • Lane 10: Cut EYFP B1, X + P
    • Lane 11: Uncut EYFP B1
    • Lane 12: Cut EYFP B2, E + S
    • Lane 13: Cut EYFP B2, X + P
    • Lane 14: Uncut EYFP B2
    • Lane 15: empty
    • Lane 16: empty
    • Lane 17: empty
    • Lane 18: empty
    • Lane 19: empty
    • Lane 20: Ladder
• Continued with gel extraction, Nanodrop, ligation, and transformation.

WEEK 11: 8/15-8/19/2011

• All ligations/transformations worked except for ECFP + B0015 and EYFP + B0015. Overnight cultures were made for all successful transformations.
• Minipreps were done and Nanodrop followed. Nanodrop yielded decent results (values ranging from 21.2-53.4 ng/uL).
• 08/17-08/21: out of Boston.

WEEK 12: 8/22-8/26/2011

• Restriction digest was performed on all ECFP and EYFP plus RBS combinations. Terminator and B0034 were also cut.
• All samples were run on a gel. All bands appeared to be cut.
  • 1st 12 well:
    • Lane 1: Ladder
    • Lane 2: Cut ECFP + J61100C
    • Lane 3: Uncut ECFP + J61100C
    • Lane 4: Cut ECFP + J61127A
    • Lane 5: Uncut ECFP + J61127A
    • Lane 6: Cut EYFP + J61100C
    • Lane 7: Uncut EYFP + J61100C
    • Lane 8: Cut EYFP + J61127C
    • Lane 9: Uncut EYFP + J61127C
    • Lane 10: empty
    • Lane 11: empty
    • Lane 12: Ladder

2nd 12 well:

• • • Lane 1: Ladder
    • Lane 2: Cut B0034
    • Lane 3: Uncut B0034
    • Lane 4: Cut B0015
    • Lane 5: Uncut B0015
    • Lane 6: empty
    • Lane 7: empty
    • Lane 8: empty
    • Lane 9: empty
    • Lane 10: empty
    • Lane 11: empty
    • Lane 12: empty
• Gel extraction and Nanodrop followed.
• All samples were then ligated with terminator and transformed. Previously cut EYFP and ECFP were ligated with B0034 and transformed.