Team:USTC/Notebook/5 1
Notebook
Together we stand
Performers
Date: May 1st
Recorder: Kaiyue Ma
Prepare solid LB culture medium as following protocol
| Component | Volume |
|---|---|
| typtone | 5g |
| yeast extract | 2.5g |
| NaCl | 5g |
| Agar A | 7.5g |
| deionized water | 500mL |
alter pH to 7.0 with NaOH
sterilized with high temperature and pressure
Prepare liquid LB culture medium as following protocol
| Component | Volume |
|---|---|
| typtone | 5g |
| yeast extract | 2.5g |
| NaCl | 5g |
| deionized water | 500mL |
alter pH to 7.0 with NaOH
sterilized with high temperature and pressure
Transformation of pSB1C3
procedure:
(1)Take the competent bacteria from refrigerator and incubate them into ice about 10 mins until it is dissolved
(2)Absorb 1 uL plasmid and mix it with bacteria solution thoroughly.
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 45 sec
(5)Put the tubes on the ice about 3 mins again.
(6)Add 900 ul LB medium into EP tubes and cultivate the bacteria at 37 degree centrigrade about 60 min.
(7)Centrifuge them at 12,000 rpm about 15 sec and we will se sediment in the tubes.
(8)Discard the supernatant liquid and leave about 220ul medium.
(9)Coat plate: add 200ul solution in a plate and 20ul to another
and then cultivate them overnight
Date: May 2nd
Recorder: Kaiyue Ma
No colonies showed.
Yesterday's experiment failed.
Date: May 4th
Recorder: Xingfu Qin
Prepare solid LB culture medium as following protocol
| Component | Volume |
|---|---|
| typtone | 1g |
| yeast extract | 0.5g |
| NaCl | 1g |
| Agar A | 1g |
| deionized water | 100mL |
autoclave the medium at 120℃ for 20 minutes
Add 100uL chloramphenicol(100mg/mL) in to the solid LB medium when its temperature down to about 60℃,then subpackage the medium into 5 plate(sterilized)
Prepare liquid LB culture medium as following protocol
| Component | Volume |
|---|---|
| typtone | 2g |
| yeast extract | 1g |
| NaCl | 2g |
| deionized water | 200mL |
subpackage the medium into 40 test tubes (5mL per tube), then autoclave the medium at 120℃ for 20 minutes
Transformation of pSB1C3
experimental materials
E-coil top10 100uL（made by ourselvise)
pSB1C3 plasmid 1uL
procedure:
(1)Absorb 1 uL plasmid on the .surface of bacteria solution softly
(3)Put the tubes on the ice about 30 mins.
(4)Make a heat shock at 42 degree centigrade about 90sec
(5)Put the tubes on the ice about 5 mins
(6)Add 200 ul LB medium into the tube and cultivate the bacteria at 37 degree centrigrade and shock at 180rpm/min about 60 mins.
(7)Centrifuge the tube at 4000 rpm for 2 mins make the bacteria deposit at the bottom of the tube.
(8)Discard 100uL supernatant liquid and resuspend the bacteria
(9)Coat plate: add the solution in a plate and spread it evenly as treatment group;use another blank plate as control group , then cultivate them overnight
Date: May 5th
Recorder: Xingfu Qin
Two colonies grow on the treatment group plate while no colony grow on the control group plate
the chloramphenicol concentration is a little bit high .
Clone picking
Add 1.7uL chloramphenicol (100mg/mL) in 5mL LB liquid medium (5mL per test tube)
Pick the colnes in the test tubes
Cultivate the bacteria at 37 degree centrigrade and shock at 250rpm/min overnight.
Date: May 6th
Recorder: Xingfu Qin
Culture Collection
Add 800uL bacteria solution and 400uL 50% glycerinum(sterilized) into 1.5mL tube , mix softly. Store the tube at -80℃.
Plasmid extraction
experimental materials：SanPrep plasmid miniprep purification kit
1.Pellet 5mL of bacteria culture by centrifuguation for 2min at 8000×g in a microcentrifuge.Discard the supernatant and remove any excess media.
2.Resuspend the cell pellet aompletely in 250uL of Buffer P1 by pipetting or vorexing
3.Add 250uL Buffer P2 and mix by inverting the tube 5 times and wait for 2 mins.
4.Add 350uL Buffer P3 and mix by inverting the tube 5 times .
5.Centerifuge the lysate at top speed(12000×g) in a microcentrifuge for 5 min.
6.Insert the spin column into a collection tube, carefully transfer all of the clear lysate from step 5 to spin column,Centerifuge at 8000×g for 30 s.
7.Discard the filtrate in the collection tube and add 500uLBuffer DW1 to the spin column and centerifuge at 9000×g for 30 s.
8.Discard the filtrate in the collection tube and add 500uL Wash Solution to the spin column and centerifuge at 9000×g for 30 s.Repeat this step once more.
9.Discard the filtrate and centerifuge at 9000×g for 1 min.
10.Transfer the spin column into a new microcentrifuge tube and add 50uL of Elution Solution into the column and wait for 1 min.
11.Centrifugen at top speed(12000×g）for 1 min. Store the eluted plasmid DNA at -20℃
Nanodrop
the eluted plasmid DNA 10.7ng/uL A260/280=1.93
Date: 5/8
by Li, Yuanheng
Subgrouping:
Team 1: evaluating the availability of R9
Team 2: connecting Sup35 to GFP (Output, inspired by TEAM VALENCIA 2010)
Team 3: designing genetic circuit (Input)
Date: 5/9
by Liang, Yonghao; Li, Yuanheng
Materials (Req.):
-
R9 15g
-
Saccharomices cerevisiae (From Prof. Hong's Lab)
-
Peptone
Date: May 11th
Recorder:Yuanheng Li
Culturing W303 (ΔURA3 ΔTRP ΔLEU ΔHis ΔAde):
(1) Prepare YPD Culture as following protocol
Component Volume peptone: 10 g yeast extract: 5 g D-glucose:5 g deionized water:500 mL
sterilized with high temperature and pressure.
(2) Add 4 mL YPD Culture into two test tubes.
(3) Pick W303 and add to two test tubes.
(4) Cultivate at 30.0°C and shake under 250 rpm and cultivate it for 48 to 72 hours.
Date: May 11th
Recorder: Kaiyue Ma
Transformation of J04450
experimental materials
competent E·coil BL21 100 μL（made by ourselves)
J04450-pSB1C3 plasmid 2 μL（50 pg/μL）
procedure:
- Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. Note: There should be 50 µL of DNA in each tube sent in the Kit.
- Thaw competent cells on ice. Then pre-chill by placing the tubes on ice.
- Pipet 2 µL of DNA into competent cell tube.
- Pipet 100 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!
- Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
- Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
- Add 900 µL of LB media per tube, and incubate at 37°C for 2 hours.
- Centrifuge the tube at 4000 rpm for 2 minutes, make the bacteria deposit at the bottom of the tube.
- Discard 800 μL supernatant liquid and resuspend the bacteria .
2. Coat plate: add the solution 200 μL in a plate and spread it , then cultivate it overnight.
Date: May 12th
Recorder: Kaiyue Ma
Transformation of J04450-pSB1C3 succeeded.
Colony picking
Add 1.7 μL chloramphenicol (100 mg/mL) in 5 mL LB liquid medium (5 mL*4).
Pick the colnes in the test tubes. Cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.
Date: 5/12
by Li, Yuanheng
Team 1:
-
Decide to use HPLC in order to detect the existence and purity of R9.
-
Fetch W303 from Prof. Hong's Lab.
Date: 5/13
by Li, Yuanheng
Meeting with Prof. Wen Jinlong: 1. Need a better presentation 2. Innovative but application required
Date: May 13th
Recorder: Kaiyue Ma
After 14 hours cultivation, the liquid mediums have all turned red, noting that the intensities aren't the same. We believe it results from the difference caused when we picked the colonies.
We assume that after sufficient time cultivating, the intensities may become about the same as expected.
So we choose two of the four tubes to continue cultivating(one is stronger while the other is weaker) to prove our assumption. The rest two are stored at 4°C.
After 12 more hours cultivating, both of the formerly chosen tubes turn into a deeper red, and can not be distinguished by eye.
Store them in fridge.
