2. U-87 MG culture
U-87 MG growth media: EMEM(Eagle's minimum essential medium)+10% FBS+100U/mL penicillin + 100µg/mL streptomycin
Cell Thawing
1) Thaw the cells in 37C waterbath.
-Do not thaw cells more than 2 minutes
-Do not allow water to contact the lid
2) Pre-wet the 15ml tube
3) Move cell to 15ml tube with pre-wet pipet
4) Set the media to 5 ml total volume in the centrifuge tube
5) Centrifuge 290g 5 minutes
6) Get rid of supernatant
7) Incubate cells in 5% CO2 37 C.
Subculturing
1) Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. T-75 flasks are recommended for subculturing this product.
2) Remove and discard culture medium.
3) Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
4) Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
5) Add 2.0 to 3.0 mL of complete growth medium and aspirate cells by gently pipetting
6) Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
7) Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:5 is recommended
Medium Renewal: 2 to 3 times per week
3. TA cloning A tailing
1) Resuspend gBlocks Gene Fragments in 20 µl TE (pH 8.0) as
described.
2) Add the following to a 15 μL reaction:
gBlocks® Gene Fragments 50 ng
Taq polymerase 1–3 Units
Taq polymerase buffer to [1X]
dATP to [0.05 mM ]
MgCl2 to [1.5 mM]
Nuclease-free H2O to final 15 μL
Total volume 15 μL
3) Incubate at 70°C for 15–30 minutes
Digestion
1) Combine the following reaction components at room temparature in the order indicated
Nuclease-free water - 15ul
10X FastDigest or 10X FastDigest Green Buffer -2ul
Plasmid - 2ul (up to 1ug)
FastDigest enzyme
2)Mix gently and spin down
3)Incubate at 37 C in a heat block or water thermostat for 5 min
Optional: Inactivate the enzyme by heating for 5 min at 65°C.
4. If the FastDigest Green Buffer was used in the reaction, load an aliquot of the reaction mixture directly on a gel.
T tailing
1)Match the following conditions
plasmid DNA – X
10X Terminal Transferase Reaction Buffer – 7.5 μl
1 mM ddTTP – 1.5 μl
CoCl2 – 7.5 μl
Terminal Transferase – 6 μl
H2O – X
DNA and water volume alignment to ensure total reaction volume 75ul
2) Incubate the reaction at 37 °C for 1.5 hour
Ligation
1) Prepare a reaction mix by adding the following reagents to a clean microcentrifuge tube
Nuclease-free water - As required to reach final reaction volume
Linearized vector DNA - 10-100ng
DNA insert - 3:1 molar excess over vector DNA
Anza T4 DNA Ligase Master Mix 5ul
Final reaction volume 20ul
2)Mix reagents by pipetting up and down
3)Incubate at room temparature for 15 minutes
4) Use 1-5ul of the ligation reaction mixture to transform competent cells
4. Competent cell
Protocol A takes much more time but is more likely to provide higher quality samples than protocol B. E.coli top10 could be used instead of DH5α.
protocol A
-materials: 50mL conical tubes, 1.5mL E-tubes, distilled water, autoclave, 70% ethanol, SOB media, ice, centrifuge,
DH5α seed stock, CCMB80 buffer, freezer, vortexer, spectrophotometer % cubettes
1) Filled all apparatus with distilled water by 3/4 and autoclave to sterilize
2) Sterilize the surroundings with 70% alcohol.
3) Inoculate E.coli DH5α seed stock in 250mL SOB media and culture in 20℃ for 16 hours. Use 50mL conical tubes.
4) O.D.600 should be up to 0.3. It shouldn't be exceeded, but rather low.
5) Centrifuge E.coli with 3000g, 4℃ for 10 minutes.
6) Discard the supernatant and add 40mL CCMB80 buffer near to zero. Vortex and add 40mL CCMB80 buffer again.
7) Shake the tube several times and incubate on ice for 20 minutes.
8) Centrifuge with 3000g, 4℃ for 10 minutes.
9) Discard the supernatant and add 10mL CCMB80 buffer near to zero. Resuspend again.
10) Dilute with CCMB80 buffer so that the O.D.600 value is from 1.0 to 1.5. (Blank should be measured with CCMB80 buffer.)
11) Incubate on ice for 20 minutes.
12) Precool the E-tubes in a freezer.
13) Divide 50ML into E-tube each.
14) Store at -80℃
protocol B
-materials: DH5α seed stock, SOB media, spectrophotometer % cubettes, ice, centrifuge, glycerol, distilled water, freezer
1) Inoculate E.coli DH5α seed stock in 250mL SOB media.
2) Make sure that O.D.600 value is from 0.3~0.5
3) Culture on ice for 30 minutes.
4) Centrifuge with 4000g, 4℃ for 2 minutes.
5) Discard the supernatant and add 25mL CaCl2 (0.1M) supplied with 15~20% glycerol. Pipette to dissolve.
6) Divide 50ML into E-tube each.
7) Everything shold be cooled down beferhand in the freezer or ice. Work slowly and strile. Store at -80℃.
SOB media ingredients(per 1L): 5g yeast extract, 20g tryptone, 0.584g NaCl, 0.186g KCl, 2.4g MgSO4
SOC media ingredients(per 1L): 5g yeast extract, 20g tryptone, 0.5g NaCl
5. Transformation
-materials: distilled water, cresol red dye, ice, centrifuge, SOC media, petri dishes, E-tube, waterbath,
competent cell, LB agar, chloroamphenichol
1) Resuspend DNA in selected wells in the Distribution Kit with 10µl distilled water. Pipet up and down several times, let sit for a few minutes.
Resuspension will be red from cresol red dye.
2) Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes
(one tube for each transformation, including your control) in a floating foam tube rack.
3) Thaw competent cells on ice: This may take 10~15 minutes for a 260µl stock.
4) Pipette 50µl of competent cells into 1.5mL procooled E-tubes. One E-tube should be filled with distilled water for control. Label each tube.
5) Keep the all tubes cool with ice.
6) Pipette 1µl of resuspended DNA into E-tube. Gently pipette up and down to resuspend.
7) Pipette 1µl of control DNA into E-tube. Gently pipette up and down.
8) Close E-tubes, incubate on ice for 30 minutes.
9) Heat shock all the tubes at 42℃ for 45 seconds. Use floating lacks.
10) Incubate on ice for 5 minutes.
11) Pipette 950µl SOC media to each transformation sample. SOC should be stored at 4℃, and prewarmed in room temperature befor the experiment.
12) Incubate at 37℃ for an hour, shaking at 200~300 rpm.
13) Pipette 100µl of each transformation onto petri dishes and spread them with spreader.
14) Spin down cells at 6800g for 3 minutes and discard 800µl ofthe supernatant. Resuspend the cells in the remaining 100µl, and pipette each transformation
onto petri dishes(LB agar with chloroamphenicol). Spread them again.
15) Incubate transformations overnight(up to 18 hours) at 37℃.
16) Pick single colonies and do a colony PCR.
6. Mini-prep
-materials: transformations, GeneElute Plasmid Miniprep Kit PLN70(includes Resuspension solution, RNase A solution, Lysis solution, Neutralization solution,
Column preparation solution, Wash solution, Elution solution, GeneElute Miniprep Binding Columns, 2mL collection tubes), 95~100% ethanol,
Centrifuge, nanodrop, loop % niddles
1) Pick single colonies from petri dish and pellet cells from inoculate in the SOB media. Culture overnight.
2) Divede mdeia into 5mL tube each and centrifuge 12000g for 1 minute. Add 78µl of the RNase A solution into the resuspend solution.
3) Discard the supernatant and resuspend the bacterial bellet with 200µl of the resuspension solution.
4) Vortex for 10 seconds to resusepend the cells until homogeneous.
5) Lyse the resuspended cells by adding 200µl of the lysis solution. Do not vortex the solution and mix the contents by gentle inversion. Wait for 5 minutes.
6) When solution becomes viscous and clear, add 350µl of the neutralization solution to precipitate the cell debris. Do not allow the lysis reaction to exceed 5 minutes.
7) Gently invert the tubes for 5 times and centrifuge at 12000g or maximum speed for 10 minutes.
8) Discard the pellet. Recenttrifuge the supernatant until the liquid becomes clear.
9) Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tubes.
10) Add 500µl of the column preparation solution to each miniprep column and centrifuge at 12000g for 30 seconds to 1 minute. And discard the supernatant.
11) Transfer the cleard lysate form step 8 to the column prepared in step 10, and centrifuge at 12000g for 1 minute.
12) Discard the flow-through liquid and add 750µl of the dilluted wash solution to the column.
13) Centrifuge at 12000g for 1 minute and discard the flow-through liquid.
14) Add 100mL 95~100% ethanol into the wash solution.
15) Recentrifuge at maximum speed for 2 minutes without any additional solution or buffer to remove the excess ethanol.
16) Transfer the column to a fresh collection tube and add 100µl of Elution solution or molecular biology reagent water to the column.
17) Add 100µl of the RNase A solution.
18) Centrifuge at 12000g for 1 minute.
19) Take the elute which is ready for immediate use. If you're not planning to use it immediately, store at -20℃.
20) Before the use, do a Nanodrop to measure the purity and amount of the DNA.
7. Transfection
protocol A: Lipofectamine 2000
-materials: Lipofectamine 2000 reagent, cells, DNA solution, 24-well, Opti-MEM medium, CO2 incubator
1) Seed cells to be at least 70% confluent at transfection. Cell density should be in 0.5~2x10^6.
2) Dilute for mounts of Lipofectamine 2000 reagent in Opti-MEM Medium(50µl x 4 per well).
3) Dilute DNA in Opti-MEM medium.
4) Add diluted DNA to diluted Lipofectamine 2000 reagent with 1:1 ratio.
5) Incubate in 37℃ in CO2 incubator for 5 minutes.
6) Add 50µl of DNA-lipid complex per well. The final DNA used per well is 500ng per well and the final lipofectamine 2000 reagent amount per well is from 1.0 to 2.5µl.
7) Incubate cells for 1~3 days at 37℃. Then analyze the transfected cells with fluorescent microscope. Use GFP filter.
protocol B: Neon electroporation transfection system
-materials: Neon electroporators, cells, DNA solution
Pulse voltage: 1300V
Pulse width: 30ms
Pulse number: 1
Cell density: 5 x 10^6
Transfection efficiency: ~70%
viability: 70%
Tip type: 10µl
