Team:ICJFLS/Experiments
Key experiments protocols
Plasmid Isolation Protocol
1. The bacteria that contained target plasmid were cultivated at 37°C overnight.
2. Take out 1,500uL solution of cultivated bacteria to a 2mL micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 min at room temperature, and then the supernatant was discarded. In this tube, repeating these steps three times to increase the concentration of the bacteria.
3. Add 250uL Solution I buffer to suspend the precipitation. Complete suspension is vital for obtaining good plasmid yield by vortex or pipetting up and down.
4. Add 250uL Solution II buffer, mix gently upside down 4-6 times to make a cleared lysate. A 2-3 min incubation at room temperature may be necessary. However, this step should not be more than 5 min, and avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity.
5. Add 350uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000rpm for 10 min at room temperature.
6. Put a HiBind DNA Mini Column into a 2mL Collection Tube.
7. The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000rpm for 1 min, the filtrate is discarded.
8. Put the Column back into the Collection Tube, add 500uL HBC Buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded.
9. Put the Column back into the Collection Tube, add 700uL Washing buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded. Repeat the washing step once.
10. Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 2 min.
11. Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at12,000 rpm for 1 min. Discard the HiBind DNA Mini Column.
12. Measure concentration of extracted plasmid DNA by Nano drop 1000, and store plasmid DNA at -20°C.
Plasmid DNA Restriction Endonuclease Digestion
DNA for downstream applications is usually digested with restriction endonucleases. Type II restriction enzymes are the most widely used in molecular biology application.
The reaction volume are carried out in 20µL.
1. Add components to a clean tube in the order as follow:
1 µL DNA (1 µg/µL)
2 µL 10x buffer
1 µL each restriction enzyme (EcoR1 and Pst 1)
15 µL ddH2O
2. Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 h.
3. Stop the reaction by heat inactivation (65°C for 15 min).
4. The digested DNA is ready for use in research applications.
DNA Extraction from Agarose Gel
Buffer Preparation. Before starting, add 24mL 100% ethanol (26mL 95% ethanol) to the 6mL DNA Wash Buffer concentrate. Add 96mL 100% ethanol (104mL 95% ethanol) to the 24mL DNA Wash Buffer concentrate.DNA Wash Buffer included with D4001S and D4001T is supplied ready to use and does not require the addition of ethanol prior to use.
1. The digested DNA was run in 1% agarose gel electrophoresis.
2. Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it in to a 1.5 mL micro-centrifuge tube.
3. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100µL (mg) of agarose gel slice adding 300µL of ADB).
4. Incubate it at 56°C for 5-10 min until the gel slice is completely dissolved. Vortex the tube every 2-3 min.
5. Transfer the melted agarose solution to a Zymo-Spin™ Column in Collection Tube.
6. Centrifuge for 1 min at 12,000 rpm. The filtratewas discarded.
7. Add 500µL of DNA Wash Buffer to the column and centrifuge for 1 min. The filtrate was discarded. Repeat the wash step once.
8. Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 2 min.
9. Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2 min at room temperature.
10. Add 20µL ddH2O directly to center of the column matrix, centrifuge for 1 min to elute DNA. Ultra-pure DNA is now ready for use.
DNA Ligation Reaction
The plasmid and insert DNAs (APL1, ADG1 and GBSS1, respectively) are digested with endonucleases prior to beginning a ligation. Following gel-purification of digested plasmid and insert DNAs, DNA concentrations are measured by a spectrophotometer to determine the concentration of the purified plasmid and insert.
1. Add components to a clean tube in 20µL volume in the order as follow:
9 µL ddH2O
3 µL Promoter DNA
5 µL Plasma DNA
1 µL 10x buffer
1 µL T4 DNA Ligase
2. Mix them gently and fully, and incubate the tube at 22°C for 20 min. The ligation product is ready to use.
The Competent Cell Preparation
1. Streak the E. coli stock on a LB-agar plate. Incubate the plate at 37°C overnight.
2. Pick a single well-isolated colony and inoculate it into 3mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3. Transfer 250uL of the saturated overnight solution to a sterile 50mL polypropylene tube containing 25mL of LB medium. Incubate the E. coli at 37°C with shaking at 220 rpm until OD600 reaching 0.6. This usually takes 2.5 h. Check the OD every 30 min after 1 h to avoid overgrowth.
4. When the medium reaches an OD600 of 0.6, chill the tube on the ice for 30 min and then centrifuge at 8000rpm for 1min at 4°C, discard the supernatant.
5. Resuspend the E. coli in 2.5mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50uL per tube. Now the competent cells are ready to be transformed.
Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5mL, PEG (wt/vol, Mw 8000) 10% 1.0g, DMSO (vol/vol) 5% 500μL, and MgCl2(pH 6.5) 50mM 500μL.
Transforming the competent cells
1. Thaw TSS cells on ice.
2. Add 5uLDNA in TSS cells, pipette gently to mix (1μl of prepped plasmid is used as control).
3. Incubate it on ice for 30 min with occasional mixing.
4. Heat shock at 42°C for 90 sec.
5. After heat shock, put it on ice for 2 min, add 0.8mL LB medium into the cells.
6.Shake and incubate at 37°C for 60 min at 120 rpm.
7.Take out 100uL medium and spread them on the appropriate agar plates with certain antibiotic.
8.Incubate the plates at 37°C overnight.
PCR protocol
In the PCR protocol, reaction components are assembled as described below. The final volume should be 20µL.
In the PCR protocol, reaction components are assembled as described below. The final volume should be 20µL.
1. Thaw all reagents on ice.
2. Assemble reaction mix into 20µL volume in a thin walled 0.2mL PCR tubes as follows:
8uL ddH2O
1 µL DNA Template
0.5uL Primer F
0.5uL Prmer R
10uL 2x Taq mixture
3. Prepare negative control reaction without template DNA.Prepare positive control reaction with template of known size and appropriate primers.
4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
5. Put the tube into a PCR instrument.
6. Set up the following PCR program: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, plus a final extension at 72°C for 10 min.
7. Check the PCR product by agarose gel electrophoresis.
Purification of His/S-tagged protein
All the procedures were performed at 4°C or on ice unless otherwise specified.
1. 10mL BL21 cells transformed with a pETDuet-APL1-ADG1 or pET28a-GBSS1 were cultured at 37°C until an OD600 of approx. 0.8.
2. Add IPTG to the culture (final 0.67mM IPTG)
3. Cultivate at 30°C for 2h.
4. Collect cells in 5mL tube, centrifuge at 5000rpm at 15 min.
5. Suspend cells in 5mL of TBS, transfer the content to 1.5mL tube.
6. Collect cells at 10000rpm at 1 min. The supernatant was discarded.
7. Add 500uL of ice-cold lysis buffer to the cell pellet.
8. Sonicate cell (4°C).
9. Clarify lysate by centrifugation at to 10000rpm at 10 min.
10. After centrifugation, save 20uL of supernatant for SDS-PAGE sample.
11. Transfer supernatant to the 1.5mL tube containing pre-balanced resin bead gel.
12. Shake gently (4°C) for 30 min.
13. Centrifuge the tube 1200rpm for 30 sec. The supernatant was discarded.
14. Wash resin bead gel with washing buffer, Centrifuge the tube 1200rpm for 30 sec. The supernatant was discarded. Repeat it once.
15. Elute the protein 3 time with 20uL of elution buffer. It is ready to prepare the SDS-PAGE sample.
SDS Gel Electrophoresis
1. Make up 30 mL of running gel by adding components to a clean glass in the order as follow:
12.3 mL H2O
7.5 Ml 1.5 M Tris-HCl, pH 8.8
0.15 mL 20% (w/v) SDS
9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)
0.15 mL 10% (w/v) ammonium persulfate (APS)
0.02 mL TEMED
2. Mix the ingredients mentioned above.
3. Pour the solution quickly into a gel casting form, and leave about 2cm below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30min for the gel to polymerize completely.
4. While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follows:
3.075 mL H2O
1.25 mL 0.5 M Tris-HCl, pH 6.8
0.025 mL 20% (w/v) SDS
0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)
0.025 mL 10% (w/v) ammonium persulfate (APS)
0.005 ml TEMED
5. Mix the ingredients mentioned above.
6. Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to produce bubbles stuck underneath and allow another 30 min - 1h for complete polymerization.
7. Preparesamples: mix the protein 4:1 with the loading buffer. Heat the sample by Boiling for 5-10 min.
8. Run gel: clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.
9. Visualize the proteins using Coomassie Brilliant Blue.