Team:UIUC Illinois/Notebook

Monday 9^th June

ASDF Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2O

Autoclaved 1.5 mL Eppendorf tubes as well as LB

Poured plates of LB and chloramphenicol (CAM)

Streaked 2 plates with E. coli DguaB (pDCAF3)

Incubated in 37 degrees celsius

Aliquoted kanamycin (KAN) in four 1mL tubes

Tuesday 10^th June

ASDF Autoclaved at 1:30pm

• 0.8 grams of YNB + 156 mL H2O
• 28 grams of salts + 490 mL H2O

Used sterile filter for 1M MgSO4 and 1M CaCl2

Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli

Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture

• Labeled this "M9 media"

Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media

Placed sample in 30 degree celsius shaker for culturing

Wednesday 11^th June

ASDF Inoculated CBB1 frozen stock in 5 mL of M9 media

Placed sample in 30 degree celsius shaker

Purified pDCAF plasmid using DNA mini kit

• Plasmid concentration was 132.9 ng/uL

Friday 13^th June

ASDF Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)

Followed bacterial transformation protocol to prepare/grow culture overnight

Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days

• Previous CBB1 culture did not grow, showed slight growth
• pDCAF3 strain in M9 did grow in the 30 degree celsius shaker

Monday 16^th June

ASDF No growth in M9 by CBB1

To grow the promoter plasmid:

• 5 mL of LB + 1 uL Amp
• Label tubes
• Grow in 30 degree celsius shaker overnight

New CBB1 trials:

• 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample

Purify genomic DNA plasmid following the Wizard Protocol

• Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
• We put our 2 promoter plasmid plates in the 4 degree celsius shelf

Tuesday 17^th June

ASDF Quantified gDNA with TECAN

• Result: 5.4 ng/uL

Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker

Wednesday 18^th June

ASDF Autoclaved 50% glycerol

Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):

• CBB1 in M9 (x2 1.5 mL tubes)
• Promoter 1
• Promoter 2 (both promoter plasmids are J23114 Ampᴿ)

Purified both promoter plasmids using the purification protocol

• Stored in the -20 degree celsius freezer in the DNA box

Thursday 19^th June

ASDF Quantified promoter plasmid (J23114 #1 and #2)

#1: 342.9 ng/ul with a ratio of 1.9 #2: 373.2 ng/ul with a ratio of 1.91

Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works

• We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
• Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
• Then we will run gel electrophoresis: Concluded that the enzyme works

Cutting the promoter with restriction enzymes

• Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
• Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius

Cutting linearized backbone

• Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
• Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius