Difference between revisions of "Team:CCU Taiwan/Notebook"

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

1. Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
2. Measure Abs600 of these 1:10 diluted cultures
3. Record the data in your notebook
4. 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).
5. 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.
6. 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

1. Add all pGAPZ A_Lac1/ pGAPZ A_Px16/ pGAPZ A_Px18 (15µl) into 20µl ECOS™ 101 Competent Cells [DH5a]
2. Incubate on ice 5 minutes
3. Heat shock at 42℃ 45 second
4. Incubate on ice 5 minutes
5. Add 140µl LB
6. Incubate at 37℃ 1hr
7. Spread on LB+ Zeocin plate

Ligation

8/9
10:00-16:00
  Day 3:Cell growth, sampling, and assay

1. Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
2. Measure Abs600 of these 1:10 diluted cultures
3. Record the data in your notebook
4. 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).
5. 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.
6. Measure your samples (Abs600 and Fluorescence measurement)
7. Spread on LB+ Zeocin plate

Transformation

1. 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.
2. Incubate on ice 15min
3. Heat shock at 42℃ 45sec
4. Incubate on ice 5min
5. Add 140µl LB
6. Incubate at 37℃ 1hr
7. 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:

https://nebcloner.neb.com/#!/protocol/re/sequential-heat/AgeI,EcoRI

8/22

8/23

8/24

8/27

8/28
Transform

1. pGAPZA-1 + His4 + Px16
2. pGAPZA-1 + His4 + Px18
3. pGAPZA-1 + His4 + Lac1
   LB + zeocin

8/29

8/30
Check
pGAPZA-1 + His4
1. Digest - AgeI