Team:JNFLS/Experiments
Key experiments protocols
Plasmid DNA Extraction
1.Take out 1,500uL solution of cultivated bacteria that contained target plasmid to a 2ml micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 minute at room temperature, and then the supernatant was removed. In this tube, repeating these steps three times to increase the concentration of the bacteria.
2.Add 250uL Solution I buffer to suspend the precipitation. Suspension should be uniform, no small bacteria block left.
3.Add 250uL Solution II buffer, mix evenly upside down 4-6 times; make the cell lysis fully until the formation of bright vision. This step should not be more than 5 minutes, and avoid severe shock, so as to avoid contamination of genomic DNA.
4.Add 350uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000rpm for 10 minutes.
5.Put a HiBind DNA Mini Column into a 2mL Collection Tube.
6.The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000rpm for 1 minute, the filtrate is removed.
7.Put the Column back into the Collection Tube, add 500uL HBC Buffer, centrifuge at 12,000rpm for 1 minute, the filtrate is removed.
8.Put the Column back into the Collection Tube, add 700uL 75% ethanol, centrifuge at 12,000rpm for 1 minute, the filtrate is removed. Repeat the wash step once.
9.Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 1 minute.
10.Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2-3 minutes. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 minute. Centrifuge at12,000 rpm for 1 minute. Discard the HiBind DNA Mini Column.
11.Measure concentration of extracted plasmid DNA by nanodrop 1000, and store plasmid DNA at -20°C.
Enzymatic Digestion of Plasmid DNA
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 hour.
3.Stop the reaction by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
4.The digested DNA is ready for use in research applications.
Gel DNA Extraction
1.Buffer Preparation
2.Before starting:
3.Add 24ml 100% ethanol (26ml 95% ethanol) to the 6ml DNA Wash Buffer concentrate.
4.Add 96ml 100% ethanol (104ml 95% ethanol) to the 24ml DNA Wash Buffer concentrate.
5.DNA Wash Buffer included with D4001S and D4001T is supplied ready to use and does not require the addition of ethanol prior to use.
6.All centrifugation steps should be performed between 10,000 -16,000 xg.
7.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.
8.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).
9.Incubate it at 37-55℃for 5-10 minutes until the gel slice is completely dissolved.
10.Transfer the melted agarose solution to a Zymo-Spin™ Columnin Collection Tube.
11.Centrifuge for 30-60 seconds. Discard the filtrate.
12.Add 200µL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the filtrate. Repeat the wash step once.
13.Place the Column into a 1.5 ml tube, add ≥6µL DNA Elution Buffer or water directly to center of the column matrix, centrifuge for 30-60 seconds to elute DNA. Ultra-pure DNA is now ready for use.
Ligation
1.Add components to a clean tube in the order as follow:
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 reaction at 16°C for overnight.
Preparing the Competent Cell
1.Streak the cell stock on a LB plate. Incubate the plate at 37°C overnight.
2.Pick a single well-isolated colony and inoculate it into 5mL of LB broth solution. Incubate it at 37°C overnight (more than 16 hours) 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 cells at 37°C with shaking at 220 rpm until OD600 reaching 0.4. This usually takes 2.0-2.5 hour. Check the OD every 30 minutes after 1 hour to avoid overgrowth.
4.When the medium reaches an OD600 of 0.4, chill the tube on the ice for 30 min and then centrifuge at 8000rpm for 1minute at 4℃, discard the supernatant.
5.Resuspend the cells in 2.5mL of ice-cold TSS solution. Aliquote 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% 500μ l, and MgCl2(pH 6.5) 50mM 500μ l.
Transforming the cells
1.Add DNA (≤20uL) to ice cold 150uL competent cells. Incubate on ice for 30 minutes with occasional mixing.
2.Heat shock at 42°C for 2 minutes.
3.After heat shock, put it on ice for 2 min; Add the cells into 0.8ml LB medium.
4.Shake and incubate at 37°C for 60 minutes at 120 rpm.
5.Take out ＜200uL medium on the appropriate agar plates with certain antibiotic.
6.Incubate the plates at 37°C overnight.
PCR experiment
1.Add components to a clean tube in the order as follow:
1 µL DNA Template
0.5uL Primer F
0.5uL Prmer R
10uL 2x Taq mixture
8uL ddH2O
2.Mix them gently and fully, and put the tube into a PCR instrument.
3.Set up the following PCR program: initial denaturing at 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 second, 55°C for 30 second, and 72°C for 60 second, plus a final extension at 72°C for 10 minutes.
4.Check the PCR product by agarose gel electrophoresis.
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.Mixing the ingredients shown above.
3.Pour the solution quickly into a gel casting form, and leave about 2 centimeters below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30 minutes 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 follow:
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.Mixing the ingredients shown 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 get bubbles stuck underneath and allow another 30 minutes - 1 hour for complete polymerization.
7.Preparing Sample: mix the protein 4:1 with the sample buffer. Heat the sample by Boiling for 5-10 minutes.
8.Running 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.