Notebook
February
Molecular biology class
E-coli genomic DNA preparation
E-coli transformation
March
Instrument operation
April
Project brainstorming-Product Positioning, HGS monomer proportion
May
Culture selection-compare Yeast, E-coli, Acetobacter aceti
Gene design
June
Interlab experiment-Calbration1,2,3
July
7/3
Start interlab experiment-cell measurement
7/5
YPD formulation
7/9
Yeast(X33) culture
7/13
Fundraising briefing session
7/16
Communicate with NCKU(interlab &
project)
7/18
Communicate with BIT(project)
8/5
19:00-20:00
Day 2:
Pick 2 colonies from each of the transformation plates and inoculate in 5mL LB medium +Chloramphenicol. Grow the cells overnight (14 hours) at 37°C and 220 rpm.
8/6
10:00-18:00
Day 3: Cell growth, sampling, and assay
- Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
- Measure Abs600 of these 1:10 diluted cultures
- Record the data in your notebook
- Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
- Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.
- Measure your samples (Abs600 and Fluorescence measurement)
Digest pGAPZ A(X3)/ pUCIDT_Lac1/ pUCIDT_Px16/ pUCIDT_Px18 with AgeI,EcoRI
8/7
10:00-12:00
Fluorescence measurement
Ligation
8/8
Day 2:
Pick 2 colonies from each of the transformation plates and inoculate in 5mL LB medium +Chloramphenicol. Grow the cells overnight (16 hours) at 37°C and 220 rpm.
Transformation
- Add all pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18 (15µl) into 20µl ECOS™ 101 Competent Cells [DH5a]
- Incubate on ice 5 minutes
- Heat shock at 42℃ 45 second
- Incubate on ice 5 minutes
- Add 140µl LB
- Incubate at 37℃ 1hr
- Spread on LB+ Zeocin plate
Ligation
8/9
10:00-16:00
Day 3:Cell growth, sampling, and assay
- Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
- Measure Abs600 of these 1:10 diluted cultures
- Record the data in your notebook
- Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
- Take 500 μL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 μL samples per time point, 32 samples total). Place the samples on ice.
- Measure your samples (Abs600 and Fluorescence measurement)
- Spread on LB+ Zeocin plate
Transformation
- Add all pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18 (20µl) into 20µl Competent Cells DH5a and pGAPZ A 1µl+19µl ddH2O into 10µl Competent Cells DH5a as positive control.
- Incubate on ice 15min
- Heat shock at 42℃ 45sec
- Incubate on ice 5min
- Add 140µl LB
- Incubate at 37℃ 1hr
- Spread on LB+ Zeocin plate
8/10
10:00-16:00
Digest pUCIDT_Lac1/ pUCIDT_Px16/ pUCIDT_Px18 with AgeI,EcoRI
Gel Extraction
8/11
Ligation
8/12
Transformation
pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18
8/13
pGAPZ-A_Lac1-pGAPZ-A_Px16-pGAPZ-A_Px18-clone
20180813 PCR (pGAPZ-A_Lac1, pGAPZ-A_Px16, pGAPZ-A_Px18)
8/14
Miniprep
Result
8/15
12:00 Digest (use AgeI, EcoRI)
Protocol reference:
8/22
8/23
8/24
8/27
8/28
Transform
- pGAPZA-1 + His4 + Px16
- pGAPZA-1 + His4 + Px18
- pGAPZA-1 + His4 + Lac1
LB + zeocin
8/29
8/30
Check
pGAPZA-1 + His4
1. Digest - AgeI