Team:TJU/Notebook

Protocal

1 molecular manipulation


Our molecular cloning experiments go as follow:


Figure 1. 图要改 111



During this process,we used the following protocols:


TransT1 transformation:


Protocol:
1. Mix 50E.coli with 1plasmids, and place them in ice cold water bath for 30min.
2. Heat shock for 45s at 42°C. Put the system back on ice for 2 min.
3. Add 500 μl of LB without antibiotics. Incubate at 37°C for 30 min. The cultures are centrifuged for 1h at 200rpm.
4. Centrifuge the cultures at 4000rpm.

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Plasmids extraction

Protocol:
1. Harvest the bacterial cells by centrifugation at12000rpm for 1min.
2. Add 250μL buffer P1.
3. Add 250μL buffer P2, turn the centrifugation tube up side down mildly for 6-8 times.
4. Add 350μL buffer P3 and turn the up side down for 6-8 times immediately. Place it at 4°C
5. Column equilibration: add 500ul Buffer BL to the Spin Column CP3. Centrifuge for 1 min at
6. Centrifuge the centrifugation tube at 12000rpm for 10min. Pick 800μL supernate and add them in the Spin Column CP3, and place it at 4°C temperature for 8-10 min. Then centrifuge the system at 12000rpm for 1min.Pour the liquid back to the column CP3. Repeat the former step.
7.Add 600μl Buffer PW diluted with absolute ethanol into the column CP3 and centrifuge the system at 12000rpm for 1min. Repeat the former step.
8. Centrifuge the column CP3 at 12000rpm for 2min. Dry the column.
9. Add 50μL EB buffer into the column CP3 and centrifuge the column at 12000rpm for 2min. Collect the plasmid in the liquid.

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Restrictionenzyme digestion.


Reaction system 100μL
Restrictionenzyme EcoRI 5μL
Restrictionenzyme RstI 5μL
10xbuffer 10μL
H2O 60μL

Gel Extraction of DNA


1. Cut off the Single goal DNA band from the gel put it into the clean centrifuge tube and weigh.
2. Add same volume of Buffer PN more than the gel slice (0.1g gel account for 100μl). Incubate the mixture at 50°C for 10 min until the gel has completely melted.
3.Place a column CA2 in a provided 2 ml collection tube. Add 500μl Buffer BL to the CA2 column, and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the TIANGEN DNA column back into the same collection tube.
4. Place a column CA2 in a provided 2 ml collection tube. Add the mixture in last step to the column CA2, and place it at 4°C temperature for 8-10 min,then centrifuge at 12,000 rpm for 1min . Discard liquid and repeat the former step.
5. Add 600μl Buffer PW into the column CA2 and centrifuge at 12,000 rpm for 1min. Pour out liquid and repeat the former step once. Attention: Buffer PW have to be diluted with absolute ethanol and store at 4°C.
6. Centrifuge at 12,000 rpm for 2min. Dry the column.
7. Add 50μl Buffer EB directly into the middle of the column membrane and place it at 60°C temperature for 15 min. Centrifuge for 2 min at 12,000 rpm to elute DNA. An optional second elution will yield any residual DNA, though at a lower concentration.


Ligation DNA segments using pEasy-Blunt Cloning Kit.


Cloning system:


ddH2O pEasy vector temperature time
mf-Lon 2μL 2μL 1μL 25℃ 20min
Rcfb-E 0.5μL 3.5μL 1μL 25℃ 10min

PCR system:


1. PCR overlap System:


5xQ5 buffer 4μL
dNTP 1μL
forwardprimer 1μL
reverseprimer 1μL
DNA segments 1 0.3μL
DNA segments 2 2.1μL
Q5 enzyme 0.2μL
ddH2O 10.4μL

2. Colony PCR system


buffer 25μL
Forward primer 5μL
Reverse primer 5μL
dNTP 10μL
Taq 5μL
ddH2O/td> 200μL

3. PCR amplification system


Buffer 4.0μL
dNTP 2.0μL
Template 1.0μL
Forward primer 0.5μL
Reverse primer 0.5μL
Taq 0.5μL
ddH2O 11.5μL

PCR program:

Initial denaturation: 9
95°C for 5min
30cycles of:
95°C for 30 min
55°C for 30min (different primers different annealing temperature)
72°C for several min (“several”depends on the length of goal sequence, 1minper 1000bp)
Final extension: 72°C for 10min


Tip: this program is just a “Simple PCR Program” that is only for reference. We need to adjust our protocol to adapt to different situations so we could get what we want.


Ligation


1. Check the concentration of DNA fragments and vector which are going to be ligated.
2. Calculate the amount of part A/partB and vector added, based on the fragment length. Note that a ligation using a molar ratio of 1:3 vector to inserts.
3. Add DNA/buffer and ligase together in the EP tube.


System：


Part 1 X μL
Vector YμL
10x T4 Ligase Buffer 1.0μL
T4 Ligase 1.0μL
ddH2O Add until the total volume is 10.0μL

4. Mix the reaction by pipetting up and down gently and microfuge briefly.
5. Incubate at 22°C for 1 hour or at 16°C for overnight.


2 MFC set up


1. Preparation work.


2 MFC set up


1. Preparation work.


1.1 Preparation of electrodes.
We use carbon felt as the material of the electrodes which are cut to 1.5cm X 1.5cm dimension for anode and 1.5cm X 2cm (width X length) dimension for cathode. Steep the electrodes with acetone and 1M hydrochloric acid for 24 hours and 12 hours respectively. After this step, electrodes are fastened to the copper wire with glue.The proton exchange membrane are steeped with 1M hydrochloric acid overnight.
1.2 Assemble the chambers.
Seal the assembled electrodes to the cover of the cell. Fix anode and cathode chambers together with clips and screw down the covers with electrodes. ATTENTION: DO NOT ASSEMBLE THE PROTON EXCHANGE MEMBRANE OR THEY WOULD MELT UNDER HIGH TEMPRATURE.
1.3 Sterilization.
Autoclave to sterilize the devices and internal components at 121℃ for 20min.


2. MFC construction.


2.1 Assemble the proton exchange membrane.
2.2 Fill the chambers with reaction system.
Fill the anode chamber with anode solution. Pour 90 mL cathode medium (potassium ferricyanide) into cathode chamber.
2.3 Bacterial injection.
2.3.1 Calculate target OD600


Measure the OD600 in the flask and decide what OD600 you want in the chamber. The following equation will help you to determine how much bacterium solution you have to extract.

2.3.2 Bacterial injection
Centrifuge the bacterium solution extracted from the flask and resuspend it with anode solution and inject them into the anode chamber.
2.4Load a resistor into the circuit, and incubate the chamber at 37℃. The MFC is finished!


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