Team:OUC-China/Result/Protocol

From 2011.igem.org

Latest revision as of 04:05, 6 October 2011

Culture media
Preparation of antibiotic
Preparation of solution IPTG
Preparation of solution cresol red
Transformation
Long term bacterial preservation
Plasmid Miniprep Kit
Concentration measurement
Enzyme digestion
Ligation
PCR

Culture media

Use NaOH (1%) to adjust pH to 7.4 and high pressure sterilization at 121°C for 20min
SOC Medium

Add: water first, sodium one by one.
Total quantity: 100ml
Way to compound
Prepare 1M glucose solution: dissolve 18g glucose in 90ml ddH2O， constant volume to 100ml. Sterilize the liquid through 0.22μm filter membrane.
Add 2ml glucose (1M) into 100ml SOB medium and mix well.
Store at 4°C
        Usage: revive the competent cell after electronic transformation.
TY Medium ( for Rhizobium phaseoli and Rhizobium alfalfa)
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0147TY Medium ( for Rhizobium leguminosarum )
M9 Medium and supplement medium

To make 1L medium, you should add following materials into 750ml sterile dH2O ( <= 50°C):
1.5*M9 solution 200ml
Dissolve the following sodium in sterile dH2O to 1L
Divide into 5X200ml, sterilization at 15psi for 15min
2.2ml MgSO4(1 mol/L)
3. 20% solution of private carbon source
4. 1 mol/L CaCl2
※If necessary, we can add amino acid and vitamins into M9 Medium
※When using E.coli with T△ （lac-proAB）or proAB on F’ plasmid, add M9 as follows:
0.4% glucose (dextrose)
5mM MgSO4·7H2O
0.01% VB1
leucine: 5-10mg/100ml

Preparation of antibiotic

Principle of antibiotics
Antibiotics are extracts from metabolin of some microbe. They can restrain the growth of bacteria, mucedine, Richettsia, etc. or even kill them in a low concentration.
There are many kinds of antibiotics and dozens of them are frequently used for medicine. Different antibiotics have different principles.
There are some enzymes which can degrade antibiotics. One kind is called A, an ectoenzyme and work very fast. Others are intracellular enzymes and work slowly. Therefore the tube added antibiotics and bacteria with A can’t be waved too long.
        Some antibiotics disturb the synthetize of bacteria’s cell wall and make the bacteria swell in water and die.
        Some attack the cell membrane in bacteria.
        Some can prevent the protein synthesize in bacteria, and stop the reproduce of bacteria.
        Some can affect synthesize of ribodesose by changing inner metabolism and make bacteria (or cancer cell) be unable to copy new cell cytoplasm and finally die.

A.Ampicillin (50mg/L)
Dissolve 500mg kanamycin in enough water, then constant volume to 10ml. Stored at -20°C in small volume. Often be added on 50μg/ml into medium.
B.Chloramphenicol 25mg/ml
Dissolve 250mg chloramphenicol in exact ethno and constant volume to 10ml. Stored at -20°C in small volume. Often be added on 12.5-25μg/ml into growth medium.

C.Kanamycin 10mg/ml
Dissolve 0.5g kanamycin in water and constant volume to 10ml. Stored at -20°C in small volume. Often be added on 10-50μg/ml into growth medium.

D.Tetracycline 10mg/ml
Dissolve 100mg tetracycline sodium in water (or dissolve alkali-free tetracycline in exact ethno) and constant volume to 10ml. Packed with aluminized paper to avoid light and stored at -20°C in small volume. Often be added on 10-50μg/ml into growth medium.
※1. Use 50-60°C water heating when tetracycline cannot dissolve completely, shaking it when necessary
※2.Keep in dark place

E.Streptomycin 50mg/ml
Dissolve 250mg in exact ethno and constant volume to 10ml. Stored at -20°C in small volume. Often be added on 10-50μg/ml into growth medium.

Preparation of solution IPTG

Isopropy1 β-D-1-Thiogalactopyranoside (IPTG) dissolve in 240mg/ml (1M) and filtrate to degerm. Dilute to 1mM (1/1000) when used.

Preparation of solution cresol red

0.1% solution cresol red
0.1g cresol red dissolve in 100ml ethno
0.1g cresol red dissolve in 0.005mol/L NaOH solution and dilute to 100ml
1% solution cresol red
0.1g cresol red dissolve in 10ml ethno
0.1g cresol red dissolve in 0.005mol/L NaOH solution and dilute to 10ml

Transformation

1.Start thawing the competent cells on crushed ice.
2.Add 50 µL of thawed competent cells and then 1 - 2 µL of the re-suspended DNA to the labeled tubes. Make sure to keep the competent cells on ice.
3.Incubate the cells on ice for 30 minutes.
4.Heating shock the cells by immersion in pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
5.Incubate the cells on ice for 5 minutes.
6.Add 200μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
7.Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
8.Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
9.Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivated the antibiotic outside of the bacteria.

Long term bacterial preservation

Materials
40% glycerol solution
Cryogenic vials
Method
Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
Add 1 ml sample from the culture of bacteria to be stored.
Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
Alternatively, pipet to mix.
Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.

Notes
While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.

Plasmid Miniprep Kit

SanPrep Plasmid DNA Kit
1. Preparation
Make sure that RnaseA has been added into BufferP1
Make sure that ethno has been added into Wash Solution( stored at 4°C)
Make sure that P2 and P3 don’t have any sediment
2. Extract 1.5-5ml overnight suspension culture and centrifuge at 8000g for 2 minutes to recollect bacteria and discard culture.
3. Add 250μl BufferP1 and suspend bacteria
4. Add 250μl BufferP2, immediately overturn the tube for 5-10 times. Stay in room temperature for 2-4 minutes to split bacteria.
5. Add 350μl BufferP3. Large amount of flocks appear. Overturn the tube for 5-10 times. Be careful don’t let the flocks disperse.
6. Centrifuge at 12,000g for 5-10 minutes. Move supernatant into a absorbing column and centrifuge 8000g for 30s. Discard liquid in collection tube.
7. ( Optional) Add 500μl Buffer DW1 and centrifuge 30s at 9,000g. Discard liquid in collection tube.
8. Add 500μl Wash Solution, centrifuge at 9,000g for 30s. Discard liquid in collection tube.
9. Repeat step8
Centrifuge empty tube at 9,000g for 1min
Using a vacuum centrifuge enrichment machines concentration，Using a vacuum centrifuge enrichment machines concentration of alcohol solvent model 45 degrees 3 minutes, you can effectively remove the residual alcohol, to ensure the quality of plasmid elution.


Put absorbing tube in a new 1.4 EP tube, and add 100μl Elution Buffer at center of absorbing film. Stay at room temp for 1min. centrifuge 1min and store the DNA solution in the EP tube.
Attention Here!
Bacteria at stationary phage have the productivity of plasmid kit.
You should mix the tube exactly when added buffer, but not waiting until the last one is added.
You must not skip step10. Otherwise the residual ethno will affect the DNA productivity and following experiments.
Elution Buffer is 2.5mM Tris-HCL, pH is 8.5. Can be replaced by TE or dH2O (pH>7.0).

Concentration measurement

Please see the concrete steps on the instruction book of NANOVUE

Enzyme digestion

NEB standard Enzyme digestion:
Note:
·lay the enzyme on the ice after take it out from refrigeratory.
·enzyme is the last one to add into the system
·mix the reactant before adding enzyme. drawing-in using Auto dispenser or shoot the extine gently, then centrifugal. Forbid oscilation!
·when cutting superhelix plasmid and embedding DNA with agarose, it need more than 1 unit/μg enzyme.

Ligation

20μl reaction system:
1μl T4 DNA ligase.
2μl buffer
Carrier(>100ng)
Part (proportion is 3:1~10:1)
ddH2O make up to 20 μl.

PCR(Product of Fermentas)

Bacterial template preparing:
Dilute 14-16h cultured bacterial suspension with the ratio of 1:50 using abacterial water, or shaking culture for 2 or 3 hours to slightly turbid state
Taq enzyme system: ---For PCR Selection
DdH2O：35.5ul；
dNTP：4ul；
10×Taq (NH4)2SO4-MgCl2 buffer：5ul；
Primer1：1.5ul；
Primer2：1.5ul；
Bacterial suspention：2ul；
Taq DNA polymerase：0.5ul；

Pfu system
DdH2O：35.5ul；
dNTP：4ul；
10×Pfu buffer +MgSO4：5ul；
Primer1：1.5ul；
Primer2：1.5ul；
Backterial suspention：2ul；
Pfu DNA polymerase：0.5ul；

Taq+Pfu system---For standardization, saving Pfu, provided by advisor Tan
DdH2O：33.5ul；
dNTP：2ul；
10×Taq +(NH4)2SO4-MgCl2：5ul；
MgCl2：4ul；
Primer1：1.5ul；
Primer2：1.5ul；
Backterial suspention：2ul；
Taq DNA polymerase：0.25ul；
Pfu DNA polymerase：0.25ul；