Below are the protocols that we used in our project. Click the boxes for more detail!
- Set up PCR mixture as follow :
- Set up thermocycler for PCR reaction as follow:
- Add the other Component in the tube. If the added volume is less, pipetting up and down is necessary.
- Mix reagents in tubes by pipetting the solution up and down slowly.
- Quick spin the PCR tube to ensure the mixture is in the bottom of the tube.
- Put it in the thermocycler (PCR machine) and set the cycle information.
- Start the cycle and wait till it is finished.
Component | Volume |
---|---|
Phusion DNA Polymerase | 0.5 μl |
5X Phusion Green HF Buffer | 10 μl |
10mM dNTPs | 1 μl |
Forward Primer | 2.5 μl |
Reverse Primer | 2.5 μl |
DNA template | 1 μl |
DNase-free ddH2O | 32.5μl |
Procedures | Temperature (°C) | Duration | Cycle |
---|---|---|---|
Initial denaturation | 98 | 30 s | 1 |
Denaturation | 98 | 5 s | 1 |
Annealing | 64 | 30 s | 25 |
Final extension | 72 | 5 mins | 1 |
Sample keping | 4 | ∞ | 1 |
- Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.
- Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber.
- Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times.
- Load your samples and appropriate marker into your wells
- Incubate at 37°C and 250 rpm for 60-120 minutes.
- Apply 130 volts to the chamber for 20 mins.
- Check your gel using a transilluminator or other UV emitting device.
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- Incubate overnight at 37°C with plates upside down.
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by trimming off extra agarose gel.
- Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (300 μl QG to 100 mg gel).
- Incubate the tube at 60°C until the gel completely dissolved.
- For DNA fragments <500 bp and >4 kb, add 1 gel volume of isopropanol to the sample and mix the reagents well.
- o bind DNA, apply 800 μl sample to the QIAquick column, and centrifuge for 1 min.
- Discard flow-through and place QIAquick column back in the same collection tube. It can be reused to reduce plastic waste.
- Add 0.75 ml of Buffer PE to wash the QIAquick column, and centrifuge for 1 min.
- Add 30 μl of distilled water to elute DNA.
- Prepare the reagents as follow:
- Pipette the solution up and down to ensure all reagents are mixed well.
- Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours.
- Purify the DNA by PCR purification kit/gel extraction kit for downstream process.
Reaction Components | Volume |
---|---|
DNA template | 0.5 μg |
10X Buffer | 2.5 μl |
Enzyme | 2 μl |
Nuclease-Free Water to final colume of: | 25 μl |
- Prepare the reaction mixture as follow:
- Allow the ligation to take place a 22-25°C for 10 minutes.
- Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional)
Reaction Components | Volume |
---|---|
DNA | 0.2M KCl/HCl |
DNA template | 10-200 ng |
10XT4 DNA Ligase Reaction Buffer | 2 μl |
T4 DNA Ligase | 1 μl |
Water | Top up to 20 μl |
- Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.
- Spread the bacterial cell (DH5α) for plasmid replication on the LB.
- Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm.
- Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h)
- Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes.
- Collect the cell by centrifuging at 1000 g for 10 min at 4°C.
- Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures!
- Collect the cell by centrifuging at 1000 g for 10 min at 4 °C.
- Centrifuge at 13,000 rpm for 1 min.
- Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer.
- Centrifuge at 13,000 rpm for 1 min.
- Column dring with centrifuge 13.000rpm for 1 min.
- Transfer all cells to one tube.
- Dispense 100 μl aliquots of competent cells into each Eppendorf.
- Store at -80 °C.
- Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.
Composition | Volume |
---|---|
0.6 M CaCl2 | 10 ml |
0.5 M PIPES pH7.0 | 2 ml |
Glycerol | 15 ml |
H2O | 73 ml |
Total | 100 ml |
- Thaw 100µL competent E. coli cells on ice for 10 minutes
- Add:
- 10-20 µl DNA from a ligation reaction mix OR
- 50-100ng DNA of a known plasmid
- Place the mixture on ice for 15 minutes.
- Heat shock at exactly 42°C for 90 seconds.
- Place on ice for 5 minutes.
- Pipette 900 µl of room temperature SOC or LB media into the mixture.
- Incubate at 37°C and 250 rpm for 60-120 minutes.
- Pellet cells at 13000rpm for 1 minutes
- Remove and dispense 600 µl of supernatant
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- Incubate overnight at 37°C with plates upside down.
- Harvest 3 mL cells at 1OD.
- Centrifuge at 14,000 rpm for 30 s.
- Discard the supernatant and resuspend the pellet with remnant.
- Resuspend with 250 μl Resuspension Buffer.
- Add 250 μl Lysis Buffer.
- Add 350 μl Neutralization Buffer.
- Centrifuge at 13,000 rpm for 10 mins.
- Transfer supernatent to column.
- Centrifuge at 13,000 rpm for 1 min.
- Add 700 μl Washing Buffer B.
- Centrifuge at 13,000 rpm for 1 min.
- Column dring with centrifuge 13.000rpm for 1 min.
- Inoculate with 30 μl distilled water.
- Centrifuge at 13,000 rpm for 1 min.
LB Broth
Formulation per one liter
- 10 g SELECT Peptone 140
- 5 g SELECT Yeast Extract
- 5 g Sodium Chloride
- 10 g SELECT Peptone 140
- 5 g SELECT Yeast Extract
- 5 g Sodium Chloride
- 12 g SELECT Agar
- 16.0 gTryptone
- 10 g Yeast Extract
- 5 g Sodium Chloride Final pH 6.8 ± 0.2 at 25°C
- Set up the following reactions:
- Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube.
- Incubate the reactions at 37°C for 60 minutes.
- Stop the reactions by placing the tubes in an ice bath for 5 minutes.
- Analyze the results of the reaction.
Component | Volume |
---|---|
DNA template | ≤2 μg |
Amino a cid Mixture (-Met) | 5 μl |
S30 Pewmix without amino acids | 20 μl |
[35S]Methionine | 1 μl |
Reverse Primer | 2.5 μl |
S30 Extract, Circular | 15 μl |
Nucelase-Free Water to final volume of : | 50 μl |
Materials:
- 10X TE Buffer
- 1M Tris HCl Buffer
- Protein lysis buffer (PLB): 50 mM Tris (pH 7.5), 50 mM NaCl, dtt. sterilized by autoclaving
- Start buffer: 20 mM Tris-HCl, pH 8.0 (At least 1 pH unit above the pI of substance)
- Elution Buffer (20mM Tris-HCl, pH 8.0, 1 M NaCl).
- Binding buffer: 4 M ammonium sulfate 132.14 g/mol in TE (Tris- EDTA)
- Wash buffer: 1.3 M ammonium sulfate in TE
- Equilibrating buffer: 2 M ammonium sulfate in TE
- Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter.
- 1% Inoculation in two 1L conical flask, each with 250 ml 2XYT solution 1x antibiotics overnight.
- Spin down 100ml cells in 50 ml falcon. Discard the supernatant.
- Wash cell pellet with 40 ml cool TE buffer.
- Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB).
- Sonicate on ice.
- Spin at 4°C at 13000 rpm for 5 min
- Collect the supernatant and dialysis overnight.
- Pipette 900 µl of room temperature SOC or LB media into the mixture.
- Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0)
- Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column.
- Trace the proteins by naked eyes.
- Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.
- Purify the proteins by HIC column followed by Ion exchange chromatography. Wash the column with 3 mL of Equilibrium buffer after HIC matrix resuspension.
- Remove the female luer for buffer running through the column to the waste collection tube.
- Mix 1 volume of protein sample to 1 volume of Binding Buffer (~200 µl).
- Transfer all 400 µl of protein/Binding Buffer mix to the column carefully without disturbing the top of the bed containing the settled HIC matrix.
- Trace the fluorescent protein by naked eyes or blue light.
- Wash the resin with 1 ml Wash buffer when the meniscus reaches the top of the bed and the run-through is collected in the waste collection tube.
- Add 1 ml TE buffer to the resin and the run-through without fluorescent proteins is collected in the waste collection tube.
- Continue adding TE buffer to run through fluorescent proteins and collect with Eppendorf.
- Run SDS-PAGE to determine purity.
- Determine protein concentration by refractometer.
- Prepare the buffers as follow:
- Diluted into buffers ranging in pH from 2-12 in 96-well plates.
- Determine absorbance/ fluorescence by Plate reader.
pH | Composition |
---|---|
2 | 0.2M KCl/HCl |
4 | 3M acetate buffer |
6 | 0.5M MES buffer |
7 | 1M PIPES buffer |
8 | 1M HEPES buffer |
10 | 0.2M NaHCO3/NaOH |
12 | 0.2M KCl/NaOH |