Revision as of 07:34, 5 October 2018

protocal

Transient transfection of Jurkat T cells

1. Cturing of cells before nucleofection. Cells shod be nucleofected after reaching 70-85% confluency, higher cell densities may cause lower nucleofection efficiencies, mycoplasma contamination will negatively influence experiments rests.
2. Combine the cells of interest, DNA and the appropriate cell-type specific Nucleofection Solution (Here Buffer C), and transfer to an amaxa certified cuvette (invitrogen cuvette). 1X106-5-106 cells, 1-5ug plasmid DNA in 1-5 H2O or TE, and 100 room temperature Buffer are used in this step. The quapty and the concentration of DNA used for nucleofection plays a central role for the efficiency of gene transfer. We strongly recommend the use of high quapty products for plasmid purification pke Qiagen endofree plasmid kit. The purified DNA shod be resuspended in deionised water or TE buffer with a concentration between 1-5ug/. The ration of A260;A280 shod be at least 1.8 for nucleofection
3. Insert the cuvette into the Nucleofector TM and choose the cell-type specific program. Press the start button "X". If the program is unknown, please following the recommend above. To avoid damage to the cells remove the sample from the cuvette immediately after the program has finished (display showing "OK")
4. Rinse the cuvette with cture medium and transfer the cells into the cture dish.
5. Incubate at 37°C until use.

Lipofectamine 2000 DNA Transfection Reagent Protocol

1、Seed cells to be 70–90% confluent at transfection.
2、Dilute four amounts of ppofectamine® Reagent in Opti-MEM® Medium Opti-MEM® Medium.
3、Dilute DNA in Opti-MEM® Medium.
4、Add diluted DNA to diluted ppofectamine® 2000 Reagent (1:1 ratio)
5、Incubate for 20 minutes at room temperature.
5、Add DNA-ppid complex to cells.
6、Incubate cells for 1–3 days at 37°C. Then analyze transfected cells.

Calcium Phosphate Transfection

Calcium Phosphate Transfection
2X HBS For 1 pter
50mM HEPES, pH 7.05 11.92 g
10mM KCI 745.60 mg
12mM dextrose 2 16 g
280mM NaCl 16.36 g
1.5mM Na2PO4 10 ml from 100x+ 40.2g Na2PO4 7xH2O/L
Dissolve, in 950 ml H2O. Adjust pH to exactly 7.5 with 1N HCL, bring up to 1L with ddH2O. Filter using 0.22 micron filter. Apquot in 50 ml Falcon tubes. Store at -20℃
2M CaCl2: Filters steripze. Store in 1 ml apquots at -20℃. Stable at RT

Transfection

1. Twenty-tour hours prior to transfection, inocate 1-2×106 cells/10 cm plate in 10 ml DMEM/F12 media + 10% BCS supplemented with L-glutamine and Pen/Strep. At the time of transfection, cells shod be about 60-70% confluent
2. The following day (20-24 hours later), transfect cells. Up to 15 to 20 µg DNA can be used for one 10-cm plate. If more than one plasmid is used for transfecting the same plate, equapze DNA amounts among different plates by adding vector plasmid DNA to some plates, so that the total amount of plasmids used for each plate is the same. For transfection.
a. Prepare the following mix for 10 cm plate.
61 vL 2M CaCl2.
10ug DNA total
Add ddH2O to 500
Vortex to mix.
b. Slowly (drop-wise) add 0.5 ml of 2x HBS while typing the mixture prepared in (a)
c Drop total 1 mI of the final mix around the plate, then mix gently. Place the plate back in the incubator.
3. Twelve to 16 hours after transfection, gently aspirate the medium and add 10 ml of pre-warmed fresh medium to the plates. Try not to disturb the fine DNA-CaPO4 precipitates on the bottom of the plate
4. The following day, harvest the cells and extract protein as needed

RT-qPCR

Extraction of RNA

1. Suck and discard DMEM cture solution. Wash cells with 500 PBS, suck and discard PBS. Add 300μl trypsin to digest the cells. Add the same amount of 10 % DMEM cture solution to stop the digestion. Transfer the cell suspension to a 1.5 ml EP tube and centrifuged at 1000 rpm/min for 5 min using a centrifuge. Suck and discard the supernatant , and add 600μl PBS to resuspend the cells. Add 500μl trizol to 170μl cell suspension, mix well, transfer the mixture to 1.5 ml EP tube, and let stand at room temperature for 10 min.
2. Add 1 / 5 volume of chloroform, shake violently, mix well, and let stand at room temperature for 10 min.
3. Pre-cool the centrifuge to 4 ℃ and centrifuge at 12000 g and 4 ℃ for 20min.
4. Transfer 250 μ L of supernatant to a new EP tube and add isopropanol of equal volume. Let stand at room temperature for 10 min and precipitate at - 20 ℃ for 1h.
5. Centrifuge at 12000 g and 4 ℃ for 20min, discard supernatant, and retain precipitate
6. Add 1 ml of 75 % ethanol prepared with DPEC water to the precipitate, wash the precipitate, centrifuge at 12000 g and 4 ℃ for 15 min
7. Discard the supernatant, keep the precipitate, and dry the EP tube upside down on the table for 30 min
8. Add 10μ LDPEC water to each tube, measure the concentration and store at - 80 ℃ for later use.

RT-PCR

1. Use takara reverse transcription kit to configure the reverse transcription system according to the following system
AMV 0.5
5×Buffer 2μl
dNTP 1μl
DPEC Water 3.75 μ L
OpgodT 0.5μl
RRI 0.25μl
The extracted RNA template 2μ L
2. Set a program of 16 ℃ ( 10 min ), 42 ℃ ( 60 min ), 85 ℃ ( 5 min ) and 4 ℃ in the PCR instrument, and put the configured system into the PCR instrument for reaction.

qPCR

1. The qPCR system was configured in a 96 - well PCR plate using Takara kit according to the following system
10×Buffer 2μl
dNTP 0.4μl
MgCl2 1.2μl
rTaq 0.3μl
Evagreen 1μl
ddH2O 13.5μl
Forward primer 0.3μ L
Reverse primer 0.3μ L
cDNA template 1μ L
2. Set up and run the following programs in QPCR
Stage1 Reps:1 95℃ 5min
Stage2 Reps:40 95℃ 15s
55℃ 30s
72℃ 30s
Stage3 Reps:1 95℃ 15s
60℃ 1min
95℃ 15s
60℃ 15s
3. Export data

Western blot

Preparation of lysate from cell culture

1. Suck and discard DMEM culture solution. Wash cells with 500ul PBS, suck and discard PBS. Add 300μl trypsin to digest the cells. Add the same amount of 10 % DMEM culture solution to stop the digestion. Transfer the cell suspension to a 1.5 ml EP tube and centrifuged at 1000 rpm/min for 5 min using a centrifuge. Suck and discard the supernatant , and add 600μl PBS to resuspend the cells. Use 380μl of them for WB.
2. Drain the PBS, then add 30μl ice-cold RIPA (0.5 ml per 107 cells/100mm dish/150cm2 flask; 0.2ml per 5x106 cells/60mm dish/75cm2 flask; 0.05ml per well/6-well dish).
3. Maintain constant agitation for 30 minutes at 4°C.
4. Centrifuge in a microcentrifuge at 4°C. 20 minutes at 12,000 rpm.
5. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.
6. Add loading buffer (5% SDS) to supernatant by volume ratio of 1:4 (50ml/200ml) then boil at 100℃ for around 5-10 minutes.

Running the gel

1. Wash glass plates and spacers
2. Place rubber seal in bottom of gel kit
3. Put the spacers in between the two pieces of glass, making sure they are level.
4. Add the clamps and plugs
5. Dispense the separation gel. Shake well immediately after adding TEMED to fill the gel. When filling the gel, 5 ml of gel can be sucked out along the glass by the gun, and the gel surface can be raised to a height of 1 cm under the rubber comb. Then add a layer of ethanol to the gel, and the gelation after liquid sealing is faster.
6. placed at room temperature for 30min (20min in summer) to be solidified, when there is a line of refraction between ethanol and gel, the gel has been condensed. When the gel is fully solidified, the upper layer of ethanol can be poured off and the ethanol is blotted dry with absorbent paper.
7. Dispense 5% concentrated gel. Immediately after adding TEMED, the mixture can be filled. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the gel, the gel should also flow down along the glass plate to avoid bubbles in the gel. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the gel solidifies, the loading volume of the sample hole is reduced, so the gel is often applied on both sides during the solidification process of the concentrated gel. After the gel has solidified, pinch the sides of the comb and pull them straight up.
8. Make up running buffer (1 in 10 dilution of stock). Need around a liter.
9. Remove comb and place gel in opposite side of apparatus.
10. load samples and molecular weight markers. 20-30 ug of total protein from cell lysate
11. Put on grey plastic top (make sure rubber seal is in place)
12. Add the plugs
13. Slowly add running buffer
14. Put on front plastic cover and run at 70V for around 30 minutes. After the sample reaches the concentrated gel, run at 120v for 1 hour.
Note: The gel percentage required is dependent on the size of your protein of interest: Protein size Gel percentage
4–40kDa 20%
12–45kDa 15%
10–70kDa 12.5%
15–100kDa 10%
25–100kDa 8%
Gradient gels can also be used.

Transferring the protein from the gel to the membrane

1. Need 6 pieces of filter paper (11 x 14.5 cm) and 1 piece of Hybond-P PVDF membrane of the same size.
2. Hydrate Hybond-P by placing in methanol for 3 minutes and in transfer buffer for 10 min
3. Remove gel from electrophoresis apparatus and cut to size
4. Place gel in transfer buffer for 10 min
5. Assemble the transfer cassette as follows (black side down): fiber pad (pre-wet in transfer buffer), filter paper (x3, pre-wet), gel (no bubbles), Hybond-P PVDF membrane (non bubbles), filter paper (x3, pre-wet) and fiber pad (pre-wet)
6. Place cassette in transfer cell (black on black)
7. Add transfer buffer to the electrophoresis tank
8. Connect to powerpac and run at constant amps (300mA) for 90 minutes, keep the temperature at 4℃
9. Remove membrance and, if necessary, keep hydrated in TBS in the cold room until needed.

Blocking and Antibody staining and Development

1. Block the membrane for 1h at room temperature or overnight at 4°C using blocking buffer(5% skim milk powder).
2. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer. We recommend overnight incubation at 4°C.
3. Wash the membrane in three washes of TBST, 5 min each.
4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1h.
5. Wash the membrane in three washes of TBST, 5 min each.
6. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
7. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

PCR(using Phanta Max Super-Fidepty DNA Polymerase PCR amppfication system)

Reagent Volume/μL
ddH2O Up to 50
2× Phanta Max Buffer 25
dNTP Mix(10 mM each) 1
primer F(10 μM) 2
primer R(10μM) 2
Phanta Max Super-Fidepty DNA Polymerase 1
template DNA X

Thermocycpng conditions for a Routine PCR:

Step Temperature (℃) Time cycles
Initial denaturation 95 30 seconds/3 min 1
denaturation 95 15 seconds 25-35
anneapng 56-72 15 seconds
extension 72 30-60 sec/kb
Final extension 72 2-5 minites 1
Hold 4 Indefinitely 1
Methods:
1. Prepare the reaction system in a PCR tube on ice, thaw the components and mix well. After use, put them back in -20 ℃.
2. Gently centrifuge to collect the pquid at the bottom of the tube.
3. Transfer the PCR tube to the PCR machine, set the parameters and start the thermal cycle.

Agarose Gel Electrophoresis

Methods(for a 1% Gel): 1. Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.
2. Measure 0.5g agarose , put it in a 250 mL Erlenmeyer flask, add 50 mL 1 x TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.
3. Add 5μL GelRed to the solution.
4. Pour the solution into the gel casting tray.
5. After the gel cools to sopd, pl out the comb.
6. Place the gel in the electrophoresis chamber with enough TAE buffer.
7. Add 10×loading buffer to the sample and mix, then transfer the mixture to the well on the gel with a pipette.
8. Power on,run at 120 V for half an hour.

Gel Extraction

According to the E.Z.N.A. Gel Extraction kit
1.Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended 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, carefly excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel spce by removing extra agarose.
3.Determine the appropriate volume of the gel spce by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel spce of mass 0.3 g will have a volume of 0.3 mL. 4. Add 1 volume Binding Buffer (XP2). 5. Incubate at 60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
4. Add 1 volume Binding Buffer (XP2).
5.Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6.Insert a HiBind® DNA Mini Column in a 2 mL Collection Tube.
7.Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column. Centrifuge at 10,000 x g for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
8.Repeat Steps 7 until all of the sample has been transferred to the column.
9. Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
10.Add 700 μL SPW Wash Buffer. Centrifuge at maximum speed for 1 minute at room temperature. Discard the ltrate and reuse collection tube.
11.Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
12.Add 50μL deionized water directly to the center of the column membrane. Centrifuge at maximum speed for 1 minute.
13.Store DNA at -20°C.

The double enzyme digestion system

Components (50) Volume/μL
10 x buffer 5
Enzyme I 1
EnzymeⅡ 1
DNA 1-2μg
ddH2O Add to 50μL
Put the system into a 37 ° C water bath for half an hour

Ligation

Components (10) Volume/μL
T4 DNA pgase 1
10 × T4 DNA pgase Buffer 1
Plasmid Skeleton molar ratio of Vector: plasmid is 1:3
Insert Gene
Sterile water Up to 10μL
Overnight at 16℃

Chemical Transformation

1. Take competent cells (E.cop DH5α) from -80°C refrigerator and put it on ice. （Set negative control by using chemically competent E.cop cells without plasmids）
2. When the competent cells dissolve (about 10min), add 10 μL DNA pgation product or 2 μL plasmid per tube, Place the mixture on ice for 30 minutes.
3. Heat shock at 42°C for exactly 90 seconds.
4. Put the 1.5 mL tubes back on ice for 3-5 minutes.
5. Add 500 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then cture in the shaker incubator at 37°C for an hour.
6. Extract 100-200 μL bacteria pquid, spread it on LB medium with relevant antibiotic.
7. Place plates upside down and incubate at 37°C overnight.

Colony PCR

using 2×Taq master Mix/2×Hieff PCR Master Mix
Reagent Volume/μL
2× Taq master Mix/2×Hieff PCR Master Mix 10
ddH2O 7
primer F(10 μM) 1
primer R(10 μM) 1
E. cop colony 1
Thermocycpng conditions for a Routine PCR:

Step Temperature (℃) Time cycles
Initial denaturation 94 5 min 1
denaturation 94 30 seconds 25-35
anneapng 50-60 30 seconds
extension 72 30 sec/kb
Final extension 72 1minites 1
Hold 4 Indefinitely 1
If loading on a gel, don’t need to add loading buffer to the mixture because Taq master Mix/Hieff PCR Master Mix contains loading dye.

Plasmid extraction

1. Take 1-5mL bacterial solution into a centrifuge tube, centrifuge at 12,000 rpm for 1 min and remove supernatant.
2. Add 250 μL SolutionⅠ in centrifuge tube, using the pipet or vortex oscillator to suspend the cells.
3. Add 250 μL Solution II in centrifuge tube and gently fpp upside down for 6-8 times to make sure the germ is fl cracked.
4.Add 350 μL Solution III, gently fpp upside down for 6-8 times to mix until white, floccent precipitate appears and centrifuge at 12,000rpm for 10 minutes.
5. Add the supernatant to the adsorption column in step 5, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
6.Add 700 μL Wash solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
7.Add 500 μL Wash solution to the adsorption column, centrifuge at 12,000rpm for l minute, discard the filtrate and reuse collection tube.
8.Centrifuge the empty adsorption column at 12,000rpm for 2 minute to dry the column matrix. (Residual ethanol may impact downstream apppcation)
9.Transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50 -100μL Elution buffer to the center of the column membrane, let sit at room temperature for 2 minutes and centrifuge at 12,000rpm for l minute, collect the plasmid solution in the centrifuge tube.
10. Store the plasmid at -20 ° C.

LB medium(For 100mL)

Component Mass/g
Tryptone 1
Yeast Extract 0.5
NaCl 1
Sterile water to 100 mL
Autoclave at 121°C for 20 min.
(1.5g agar shod be added before autoclaving to make sopd LB)

CloneExpress

According to the CloneExpress Mtis One Step Cloning Kit
1).Preparation for the pnearized cloning vectors；
Select appropriate cloning sites, and pnearize the cloning vector.The cloning vectors can be pnearized by restriction digesting with endonuclease or by reverse PCR amppfication.
2).Design of PCR primers of the insertions；
The principle for the design of ClonExpress® MtiS primers is: introduce homologous sequences (15 bp～20 bp)into 5’ end of primers, aiming to making the ends of amppfied insertions and pnearized cloning vector identical to the ends of their neighbours which is required for recombination reaction.
3).PCR amppfication of the insertions；
Insertions can be amppfied by any polymerase (Taq DNA polymerase or other high-fidepty polymerases),but to prevent mutations introduced during PCR, high-fidepty polymerases is highly recommended.
4).Recombination reaction；
Set up the following reaction on ice. Spin briefly to bring the sample to the bottom before reacting.
ddH2O Up to 20 μl
5×CE MtiS Buffer 4 μl
pnearized cloning vector x ng
PCR products of insertions y ng
Exnase® MtiS 2 μl
5).Transformation and plating；
6)Selection of the positive colony .