Team:NAU-CHINA/Protocols

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Transient transfection of Jurkat T cells

1. Culturing of cells before nucleofection. Cells should be nucleofected after reaching 70-85% confluency, higher cell densities may cause lower nucleofection efficiencies, mycoplasma contamination will negatively influence experiments results.
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 ul H2O or TE, and 100ul room temperature Buffer are used in this step. The quality 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 quality products for plasmid purification like Qiagen endofree plasmid kit. The purified DNA should be resuspended in deionised water or TE buffer with a concentration between 1-5ug/ul. The ration of A260;A280 should 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 culture medium and transfer the cells into the culture dish.
5. Incubate at 37°C until use.
1、Seed cells to be 70–90% confluent at transfection.
2、Dilute four amounts of Lipofectamine® Reagent in Opti-MEM® Medium Opti-MEM® Medium.
3、Dilute DNA in Opti-MEM® Medium.
4、Add diluted DNA to diluted Lipofectamine® 2000 Reagent (1:1 ratio)
5、Incubate for 20 minutes at room temperature.
5、Add DNA-lipid 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 Liter
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. Aliquot in 50 ml Falcon tubes. Store at -20℃
2M CaCl2: Filters sterilize. Store in 1 ml aliquots at -20℃. Stable at RT
Transfection
1. Twenty-tour hours prior to transfection, inoculate 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 should 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, equalize 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 500ul
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 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. 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 0.5ul
5×Buffer 2μl
dNTP 1μl
DPEC Water 3.75 μ L
OligodT 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

PCR(using Phanta Max Super-Fidelity DNA Polymerase PCR amplification 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-Fidelity DNA Polymerase 1
template DNA X
Thermocycling conditions for a Routine PCR:
Step Temperature (℃) Time cycles
Initial denaturation 95 30 seconds/3 min 1
denaturation 95 15 seconds 25-35
annealing 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 liquid 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):

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, 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 1 g/mL, the volume of gel is derived as follows: a gel slice 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 (50uL) 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 (10uL) Volume/μL
T4 DNA ligase 1
10 × T4 DNA Ligase 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.coli DH5α) from -80°C refrigerator and put it on ice. （Set negative control by using chemically competent E.coli cells without plasmids）
2. When the competent cells dissolve (about 10min), add 10 μL DNA Ligation 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 culture in the shaker incubator at 37°C for an hour.
6. Extract 100-200 μL bacteria liquid, 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. coli colony 1
Thermocycling conditions for a Routine PCR:
Step Temperature (℃) Time cycles
Initial denaturation 94 5 min 1
denaturation 94 30 seconds 25-35
annealing 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

According to the Plasmid Extraction Mini kit
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 flip upside down for 6-8 times to make sure the germ is full cracked.
4.Add 350 μL Solution III, gently flip upside down for 6-8 times to mix until white, flocculent 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 application)
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 should be added before autoclaving to make solid LB)

CloneExpress

According to the CloneExpress Multis One Step Cloning Kit
1).Preparation for the linearized cloning vectors；
Select appropriate cloning sites, and linearize the cloning vector.The cloning vectors can be linearized by restriction digesting with endonuclease or by reverse PCR amplification.
2).Design of PCR primers of the insertions；
The principle for the design of ClonExpress® MultiS primers is: introduce homologous sequences (15 bp～20 bp)into 5’ end of primers, aiming to making the ends of amplified insertions and linearized cloning vector identical to the ends of their neighbours which is required for recombination reaction.
3).PCR amplification of the insertions；
Insertions can be amplified by any polymerase (Taq DNA polymerase or other high-fidelity polymerases),but to prevent mutations introduced during PCR, high-fidelity 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 MultiS Buffer 4 μl
Linearized cloning vector x ng
PCR products of insertions y ng
Exnase® MultiS 2 μl
5).Transformation and plating；
6)Selection of the positive colony .