1. Diary
2. Protocols
M2: Transformation
1) Get the competent cells from – 80℃and wait until fusion.
2) Add 2 - 3 ul of each plasmid sample or all the ligation product into the 100 µ l of competent E. coli cells. Mix and incubate on ice for 30 min.
3) Heat pulse for 90 sec, at 42℃. Put back to ice and incubate for 2min.
4) Add 800uL LB non-antibiotic liquid medium into each microcentrifuge tube Shake the microcentrifuge tubes in shaker, at 37 degree, for 30 min to recover.
5) Plate 2000 uL of the liquid medium with transformed cells immediately, on prewarmed LB antibiotic agar plates. Incubate overnight at 37 ℃.
M5: Plasmid Mini Kit I Spin
Basically base on the E.Z.N.A.™ Plasmid Mini Kit I Spin Protocol of the OMEGA.
1) Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hr at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 ml culture tube or a flask with a volume of at least 4 times the volume of the culture.
2) Decant or Pellet bacterial cells by centrifugation at 10,000 x g for 1 min at room temperature.
3) Re-suspend the bacterial pellet by adding 250 μl of Solution I/RNase A solution, and vortexing (or pipetting up and down). Complete re-suspension (no visible cell clumps) of cell pellet is vital for obtaining good yields. Transfer suspension into a new 1.5 ml microcentrifuge tube.
4) Add 250 μl of Solution II and gently mix by inverting and rotating tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Note: Do not allow the lysis reaction to proceed more than 5 min. (Store Solution II tightly capped when not in use to avoid acidification of Solution II from CO2 in the air.)
5) Add 350 μl of Solution III and mix immediately by inverting several times until a flocculent white precipitate forms. Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
6) Centrifuge at 13,000 x g for 10 min at room temperature. Promptly proceed to the next step.
7) Prepare a HiBind DNA Mini Column by placing into a 2 mL collection tube. Add 100 μl of Equilibration Buffer. Centrifuge at 13,000 x g for 30-60 seconds. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
8) Add the cleared supernatant from step 6 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Ensure that the pellet is not disturbed and that no cellular debris has carried over into the HiBind DNA Mini Column. Centrifuge at 13,000 x g for 1 min at room temperature to completely pass lysate through the HiBind DNA Miniprep Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
9) Add 500 μl of HB Buffer and centrifuge at 13,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA Mini Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube. This step ensures that residual protein contaminations are removed, thus ensuring high quality DNA that will be suitable for downstream applications.
10) Add 700 μl of DNA Wash Buffer (diluted with absolute ethanol) and centrifuge at 13,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA Column. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
11) Repeat wash step 10 with another 700 μl of DNA Wash Buffer (diluted with absolute ethanol).
12) Centrifuge the empty HiBind Mini Column at 13,000 x g for 2 min to dry the column
13) Place the HiBind DNA Mini Column into a new/clean 1.5 ml microcentrifuge tube. Depending on desire concentration of final product, add 30-100 μl 60℃ ddh2o and incubate at room temperature for 5 minute. Centrifuge for at 13,000 x g for 1 min to elute DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
M6-7: Agarose gel electrophoresis
1%~2% Agarose and 1%TAE
1) Test gel: 120V, 25~30 min.
2) Collect gel: 80V, 55~60 min.
M9-11: PCR
1.1) Takara rTaq®
Program:
1 94℃ 5’
2 94℃ 30’’
3 Based on the temperature of primers 30’’
4 72℃ Based on the length (1Kb/min)
5 72℃ 5’
6 4℃ -/-
7 END
2~4 Perform 30cycle
1.2) Takara PrimeStar®
Program:
1 94℃ 5’
2 94℃ 30’’
3 Based on the temperature of primers 30’’
4 72℃ Based on the length (1Kb/min)
5 72℃ 5’
6 4℃ -/-
7 END
2~4 Perform 30 or 35cycle
M12: Digestion (TAKARA Restrictive Enzymes)
Condition: DNA, 37℃ for 2~2.5h; Plasmids, 37℃ for 4.5~5h
M13: Ligation (NEB T4 Ligase)
Note: The units of gene and plasmid ligated in 20μl system are based on their relative concentration in the water, or the relative intensity of brightness on the gel, which has formula as: 3 = (ax/m) / (by/n). Where a and b are the relative intensity of brightness, m and n are lengths of gene and plasmid, x and y are gene and plasmid, respectively.
M14: Gel Extraction
Basically base on the E.Z.N.A.™ Gel Extraction Spin Protocol of the OMEGA.
1) Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended however, that fresh TAE buffer, or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
2) When adequate separation of bands has occurred, CAREFULLY excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3) Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a density of 1g/ml of gel, the volume of gel is derived as follows: a gel slice of mass 0.3g will have a volume of 0.3 ml. Add an equal volume of Binding Buffer(XP2). Incubate the mixture at 60℃ for 10 min or until the gel has completely melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.
4) Place a HiBind DNA Mini Column in a provided 2 ml collection tube.
5) Apply 700µl of the DNA/agarose solution to the HiBind DNA Mini Column, and centrifuge at 10, 000 x g for 1 min at room temperature.
6) Discard liquid and place the HiBind DNA Mini Column back into the same collection tube. For volumes greater than 700µl, load the column and centrifuge successively, 700 µl at a time. Each HiBind DNA Mini Column has a total capacity of 25 µg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.
7) Add 300 µl of Binding Buffer (XP2) into the HiBind DNA Mini Column. Centrifuge at 10,000 x g for 30 to 60 seconds at room temperature to wash the HiBind DNA Mini Column. Discard the flow-through and re-use the collection tube.
8) Wash the HiBind DNA Mini Column by adding 700µl of SPW Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000 x g for 30 to 60 seconds at room temp.NOTE: SPW Wash Buffer Concentrate must be diluted with absolute ethanol before use.
9) OPTIONAL: repeat step 8 with another 700µl of SPW Wash Buffer diluted with absolute ethanol. NOTE: Perform the second wash step for any salt sensitive downstream applications
10) Discard liquid and centrifuge the empty HiBind DNA Mini Column for 2 min at maximal speed ( 13,000 x g) to dry the column matrix.
11) Place a HiBind DNA Mini Column into a clean 1.5 ml microcentrifuge E.Z.N.A.™ Gel Extraction Kit Manual tube. Add 30-50 µl 60℃ ddh2o and incubate at room temperature for 5 minute. Centrifuge for 1 min at maximal speed ($13,000 x g) to elute DNA.
12) Yield and quality of DNA: Determine the absorbance of an appropriate dilution of the sample at 260 nm and then at 280 nm.
M16: Freeze-Thaw DNA Extraction
1) Dissolve the bacteria in ddwater
2) Put the sample in -80℃ for 5min, then move to 100℃ for 5min
3) Repeat step 1 again
4) Put sample in -80℃ for 5min again, than melt in 37℃ for 10min
M: Prescription of usual reagents and mediums
1) Agarose of DNA electrophoresis: 1% agarose.
2) Buffer of agarose gel electrophoresis (50×TAE): 2M Tris-acetic acid, 100mM EDTA.
3) LB liquid medium: 10g NaCl, 10g tryptone and 5g yeast extract in 1L distilled water.
4) LB solid medium: 10g NaCl, 10g tryptone, 5g yeast extract and 18g agar in 1L distilled water.
5) IPTG: 24mg/mL IPTG.
6) Antibiotics: 100mg/mL Ampicilin, 50mg/mL kanamycini, 50 mg/mL chloramphenicol, 50μg/mL
7) Nalidixic acid.
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