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Team:Tianjin/Protocol
From 2013.igem.org
Luria Bertani Medium
| Tryptone | 10g/L |
| Yeast extract | 5g/L |
| NaCl | 10g/L |
| Solid Luria Bertani medium: Add 15g/L Agar into Luria Bertani medium | |
M9 Medium
| Na2HPO4·12H2O | 15.1 g/L |
| KH2PO4 | 3 g/L |
| NaCl | 0.5 g/L |
| NH4Cl | 2 g/L |
| MgSO4·7H2O | 0.25 g/L |
| CaCl2 | 11 mg/L |
| FeCl3 | 27 mg/L |
| ZnCl2·4H2O | 2 mg/L |
| Na2MoO4·2H2O | 2 mg/L |
| CuSO4 | 1.9 mg/L |
| H3BO3 | 0.5 mg/L |
| Thiamine | 1 mg/L |
| Bis-tris | 200 mmol/L |
| Glucose | 18g/L |
Extracting Alkanes
1. Obtain 500μl sample in 1.5 ml microcentrifuge tube from 100ml medium.
2. Add 500μl pure ethyl acetate into sample.
3. Extract by adding 0.5mL of EthylAcetate, shaking at max speed for 10 min.
4. Separate the water layer and EthylAcetate layer by centrifuge at 5000 rpm for 5min at 4℃
5. Remove 300-400μl of the top Ethyl Acetate layer, filtering by membrane, and transfer to a 1.5 ml microcentrifuge tube.
6. Store extracting sample at -20℃ bridge
Ligation
1. Check the concentration of DNA fragments and vector which are going to be ligated.
2. Calcμlate the amount of part A/partB and vector added, based on the fragment length. Note that a ligation using a molar ratio of 1:3-1:5 vector to inserts.
3. Add DNA/buffer and ligase together in the EP tube.
| Reaction system | 10.0μL |
| Part A | A.0μL |
| Vector | V.0μl |
| 10x T4 Ligase Buffer | 1.0μL |
| T4 Ligase | 0.2μL |
| Add ddH2O 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 40 min.
Restriction Enzyme Digestion
To check if the two selected restriction enzymes can perform effective catalysis in the same solution
1. Mix DNA solution with the suitable amount of the master mix.
a.
| Reaction system | 10.0μL |
| DNA solution | 6.0μL |
| 10x FD Buffer | 1.0μl |
| Each restriction enzyme | 0.2μL |
| ddH2O | 2.8μL |
| Reaction system | 30.0μL |
| DNA solution | 3.0μL |
| 10x FD Buffer | 3.0μl |
| Each restriction enzyme | 1.0μL |
| ddH2O | 23.0μL |
2. Pipette up and down in the EP tube.
3. Incubate: 37°C for 30-40 min
Competent Cell (e.g. E.coli BL 21)
1.Inoculate 5μl BL21(Glycerol Storage) into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking
2.Inoculate 50μl BL21 (from step 1) into 3 ml LB medium, inoculate a culture of 3ml LB medium, incubate for 2 h at 37℃,with 220rpm shaking
3.Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃
4.Add 1 ml pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution
5. Place the microcentrifuge tube on ice for 20min
6. Repeat step3 and step 4
7. Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃
8. Add 100 μl pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution
9. Place the microcentrifuge tube on ice for 20min
10. Store Competent Cell at -80℃ bridge
DNA Agarose Gels
1. Prepare a 1% weight-to-volume agarose gel(take 100ml as example)
2. Dilute stock of 50×TAE to 1×with ddH2O.
3. Measure 100 ml of 1×TAE buffer.
4. Transfer 1×TAE buffer to Erlenmeyer flask.
5. Weigh out enough agarose to make 1% gel. (1% of 100mL is 1.0 g)
6. Transfer agarose to Erlenmeyer flask.
7. Melt agarose in microwave, stirring every 15-20 seconds until completely melted.
8. Allow gel to cool until Erlenmeyer flask can be handled comfortably. Then add 1:20 volume ratio (5μl) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.
9. Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.
Agarose Gel Electrophoresis
1. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
2. Fill the rig with 1x TAE buffer.
3. Load 5µL of DNA maker into lane.
4. Mix 1µL of 10x loading buffer with 10µL DNA sample, load them into lane.
5. Run at 150V for 30 min.
6. Use Gel imaging system check gel.
7. Take picture for gel.
PCR Purification
TIAN™quick Midi Purification Kit
1. Balance the absorption column
a. Put the absorption column CB2 into the collection tube. Add 500 µl of the buffer BL to the absorption column CB2 and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the column back into the same collection tube.
2. Add the buffer PB
b. Determine the appropriate volume of the PCR reaction mixture.
c. Add 5 times the volume of the buffer PB to the mixture and then mix by shaking or overtaxing the tube in increments.
3. Absorption
d. Apply the mixture to the column. For volumes greater than 800 µl, load the column and centrifuge successively, 800 μl at a time.
e. Place the column at -20℃ for 5 min then centrifuge at 12,000 rpm for 1 min at room temperature.
f. Apply the mixture in the collection tube to the column and then repeat step "e". Discard liquid and place the column back into the same collection tube.
4. Wash
g. Wash the column by adding 600μl of PW Wash Buffer diluted with absolute ethanol. Centrifuge at 12,000 rpm for 1 min at room temp.
Note: PW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, PW Wash Buffer must be brought to room temperature before use.
h. Repeat step "g" with another 600μl of PW Wash Buffer diluted with absolute ethanol.
I. Discard liquid and centrifuge the empty column for 2 min at 12,000 rpm to dry the column. Do not skip this step, it is critical for the removal of ethanol from the column.
j. Place a column into a clean microcentrifuge tube. Then place the tube into the drying baker for 10min at 50℃ to dry the column matrix.
5. Elution
k. Add 50-70μl（depending on desired concentration of final product） of EB Buffer directly onto the column matrix.
l. Incubate at 50℃ for 2 min. Centrifuge for 2 min at 12,000 rpm to elute DNA.
m. Apply the mixture in the tube to the column and then repeat step "l" to yield any residual DNA.
Gel Extraction of DNA (Spin Column Extraction)
TIANgel Midi Purification Kit
1. Excise gel slice containing DNA fragment of interest.
a. Gel electrophoresis fractionates DNA fragments.
b. The gel is exposed to UV to find the DNA fragments (stained by Ethidium bromide).
c. The goal DNA band is identified.
d. Physically remove the slice of gel contains the goal DNA with clean surgical blade.
2. DNA Purification
e. Determine the appropriate volume of the gel slice by weighing it in a Clean 1.5 ml microcentrifuge tube.
f. Add 3 times volume of Buffer PN more than the gel slice (0.1g gel account for 100μl).
g. Incubate the mixture at 50°C for 10 min or until the gel has completely melted.
h. Mix by shaking or overtaxing the tube in increments of 2 minutes.
I. Place a TIANGEN® DNA column CA2 in a provided 2 ml collection tube. Apply 500μl Buffer BL to the TIANGEN® DNA 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.
j. Apply 700 μl of the DNA/agarose solution to the TIANGEN® DNA column, incubate at -25°C for 5 min and centrifuge at 12,000 rpm for 1 min at room temperature.
k. Put the liquid back into the TIANGEN® DNA column and redo j. Discard liquid and place the TIANGEN® DNA column back into the same collection tube. For volumes greater than 700 µ l, load the column and centrifuge successively, 700 μl at a time. Each TIANGEN® DNA column has a total capacity of 25μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.
l. Add 600 μl Buffer PW diluted with absolute ethanol into the TIANGEN® DNA column and incubate at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min at room temperature to wash the column. Discard the flow-through.
Note: Buffer PW Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, Buffer PW must be brought to room temperature before use.
m. Redo step l and re-use the collection tube.
n. Centrifuge the empty TIANGEN® DNA column at 12,000 rpm for 2 min to dry the column. Do not skip this step, it is critical for the removal of ethanol from the TIANGEN® DNA column.
o. Place the TANGEN® DNA column opened into a clean 1.5 ml microcentrifuge tube and incubate at 50 °C for at least 15 minute until there is no smell of ethanol.
Add 50μl Buffer EB directly into the column and incubate at 50 °C for 5 minute. Centrifuge for 2 min at 12,000 rpm to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
Error -Prone PCR
1. Make up a master mix of everything into one microcentrifuge tube.
| reaction system | 100.0μL |
| 10x error Buffer(Mg-Mn-) | 10.0μL |
| 100mM MgCl2 | 7μL |
| 10mM MnCl2 | 5μL |
| dNTP | 8μL |
| dCTP | 8μL |
| dTTP | 8μL |
| Template | 1.0μL |
| Forward primer | 3.0μL |
| Reverse primer | 3.0μL |
| enzyme | 1μL |
| ddH2O | 46μL |
2. Pipette up and down in the microcentrifuge tube, drain or50.0μL solution to each PCR tube.
3. Run the "Error-Prone PCR" program, and adjust your extension time as described below.
The "Error-Prone PCR" program
Initial denaturation: 95°C for 5min
25cycles of:
95°C for 30 min
55°C for 30min (different primers different annealing temperature)
72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)
Final extension: 72°C for 10 min
ColonyPCR
1. Make up a master mix of everything into one microcentrifuge tube.
| reaction system | 10.0μL |
| dNTP | 1.0μL |
| Taq Buffer | 1.0μL |
| up primer | 0.2μL |
| down primer | 0.2μL |
| Enzyme(fast taq) | 0.1μL |
| ddH2O | 7.5μL |
2.Run the "Simple PCR" program, and adjust your extension time as described below.
The "Simple PCR" program
Initial denaturation: 95°C for 5min
30cycles of:
95°C for 30 min
55°C for 30min (different primers different annealing temperature)
72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)
Final extension: 72°C for 10min
Fluor Spectrophotometry
1.Obtain 1000μl sample in 1.5 ml microcentrifuge tube from a 3ml overnight cultures E.coli.
2.Harvest the bacteria cells by centrifuge at 8000 rpm in 1.5 ml microcentrifuge tube for 2min.
3. Add 1ml water, mix bacteria cells by pipetting solution
4. Remove 200μL sample and add 2000μL water, measure the optical density(OD)
5. Remove μL sample and add μL water, measure the fluorescence intensity.
6. Calculate the fluorescence intensity of per OD sample, build the model
