Team:UCSF/Protocols
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(→Antibody Staining) |
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Transfected and Untransfected cells of the same cell type.<br> | Transfected and Untransfected cells of the same cell type.<br> | ||
Cells with empty vector or put through electroporation without vector<br> | Cells with empty vector or put through electroporation without vector<br> | ||
- | Dylight 488-conjugated AffiniPure Goat anti-mouse IgG, F(ab’)2, <br>Jackson Immuno Research cat# 115-485-072, Lot# 83241 <br>[should be protected from light]. | + | Dylight 488-conjugated AffiniPure Goat anti-mouse IgG, F(ab’)2, <br>Jackson Immuno Research cat# 115-485-072, Lot# 83241 <br>[should be protected from light].<br> |
- | Pierce Human IgG, whole molecule, ThermoScientific, Cat# 31154, Lot# LE1311903. | + | Pierce Human IgG, whole molecule, ThermoScientific, Cat# 31154, <br>Lot# LE1311903. (concentration=11mg/ml).<br> |
D-PBS CMF (Calcium, Magnesium free PBS).<br> | D-PBS CMF (Calcium, Magnesium free PBS).<br> | ||
Wash Buffer: 0.5% BSA, 0.1% Sodium Azide in D-PBS CMF.<br> | Wash Buffer: 0.5% BSA, 0.1% Sodium Azide in D-PBS CMF.<br> |
Revision as of 03:14, 27 October 2010
AarI Digest1. Label PCR tubes. Aar1 Digest Reagents: Note: x ul dH2O may change in volume for different reactions in order to bring total volume up to 60 ul. 3. Briefly vortex and spin down the reaction. Note: PCR program can be set to 37C for 3 hours and cooled down to 4C indefinitely.
PCR with Phusion enzyme1. Label PCR tubes. PCR Reagents 3. Vortex and spin down the reaction.
Note: Times may differ for different reactions based on the size of the desired PCR product. Annealing temperature may also differ depending on primer properties.
LigationLigation Reagents: Ligation Reaction: 2. Calculate amount of buffer needed depending on concentration for volume needed. A typical ligation reaction volume can be 20 ul. 3. Add reagents together, from smallest volumes to largest. Distilled water can be used to bring the volume up so that the reaction has the proper buffer concentration for the reaction. Ligase or enzymes in general should be added at the end. 4. Vortex or pipet up and down to mix. Spin down afterwards if vortexing. 5. Let reaction sit at room temperature for 5 to 20 minutes depending on ligase used. The ligase may start acting up if left too long.
Agarose Gel ElectrophoresisGel Electrophoresis Reagents/Materials: Preparing the agarose solution: 1. Pour in flask the amount of TAE buffer desired. This will be the volume of agarose solution made. 2. Measure out the amount of agar needed for desired concentration. For example to get a 1% agarose gel, you would add 1 gram of agarose powder to 100 ml of TAE buffer. Most concentrations commonly fall between 0.7- 2%. Higher concentrations of agarose are better suited for small fragment separation while lower concentrations are suitable for large fragments. 3. Microwave three minutes, may vary depending on volume in flask. Make sure cap is on loosely. 4. Using hot gloves, swirl bottle gently to ensure complete dissolving of agar. The solution should be clear when agar is completely dissolved. If it is not, microwave for another minute. 5. Place flask of agarose solution in 80C bath to store as to prevent solidifying of agarose solution.
1. Secure gel tray in caster 2. Pour desired volume of premade agarose solution into 50 ml conical tube. Volume desired will vary depending on gel tray size and desired thickness of gel. Thin gels solidify quicker and can show faint bands clearer, but the wells of thicker gels can hold more DNA. 3. Add Sybr safe to agarose solution. For 10 ml of agarose solution, add 1ul Sybr Safe. The Sybr safe is used to stain and visualize the DNA when viewed under a blue light. 4. Invert tube several times until Sybr safe is equally distributed. Invert gently to avoid creating air bubbles. 5. Pour contents of conical tube into secured gel tray. 6. Place gel comb into notches of gel tray. Check that comb is evenly submerged in agarose. The comb will form wells in the gel to load DNA. 7. Dry at room temperature. The gel will appear opaque when done. Colony PCRColony PCR reagents: 1. After adding reagents to PCR tube, pick a colony and touch the bottom of the PCR tube with the pipette. 2. Place PCR tubes into the PCR machine and set the cycling parameters to be optimal for the piece of DNA being copied.
Transformation of E. coliTransformation Reagents 1. Prewarm LB plates in the 37C incubator 2. Remove desired cells from -80C freezer (or other storage location) and melt over ice for at least 10 minutes. (Make sure to aliquot the correct amount of cells for transformation) 3. Pipette up 5ul of your plasmid DNA and gently pipette into the suspended cells. Do not mix by triterating the solution (pipetting up and down). 4. Let the cells sit on ice for 15-30 minutes. 5. Heat shock your cells for 75 seconds in a 42C water bath. 6. Quickly return your tubes to the ice bucket for 2-3 minutes. 7. When you are ready to plate, remove your prewarmed plate from the incubator. Using sterile technique, carefully pipette your entire tube of cells onto the LB plate. 8. Again using sterile technique, spread the cells across your plate using either sterile beads or another spreading tool. 9. Put the plate in the 37C incubator for 5-15 hours. Gel Extraction/Purification of DNAGel Extraction/Purification Materials & Reagents: 1. Using a sterile blade, carefully excise the desired band from your gel. 2. Transfer the agarose chunk into a new 2ml microcentrifuge tube. 3. Weigh the excised agarose chunk and add 3 times its weight in microliters of QG buffer. 4. Move the tube to a melting block for 10 minutes at 50C or until the agarose is completely melted. 5. Add isopropanol to the tube at a 1:3 ratio with QG, then vortex the solution briefly to help precipitate the DNA. 6. Transfer the solution to a Qiagen gel extraction/PCR purification spin column. 7. Spin for 1 minute at 13,000rpm and dump the supernatant. 8. Add 500μl of QG buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum the flowthrough. 9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum flowthrough. 10. Repeat Step 9 11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum) 12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column. 13. Place tubes in a -20oC freezer box.
MiniprepMiniprep Materials 1. Spin down bacterial culture at 3500 rpm for 5 minutes 2. Aspirate supernatant. Change tips for every sample. 3. Resuspend in 250μl of P1 Buffer. Vortex until cell pellet is not visible. Transfer into a 1.5 ml tube. 4. Add 250μl of P2 Buffer. Invert 5-6 times. Wait 3-5 minutes. 5. Add 350μl of N3 Buffer. Invert 5-6 times or until colorless 6. Centrifuge at 13,000 rpm for 10 minutes. Label blue spin columns. Transfer supernatant into blur spin columns. 7. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant. 8. Add 500μl of PB buffer. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant or vacuum supernatant. 9. Add 750μl of PE Buffer. Centrifuge columns at 13,000 rpm for 1 min and dump or vacuum supernatant. 10. Repeat Step 9 11. Centrifuge columns at 13,000 rpm for 1 min and dump supernatant. (Skip if you are using a vacuum) 12. Add 62μl of EB Buffer to spin column. Place spin column in a 1.5 ml tube. Centrifuge tune at 13,000 rpm for 1 min. Remove spin column. 13. Place tubes in a -20oC freezer box.
MaxiprepMaxiprep Materials & Reagents 1. Pick a single colony and inoculate in 2-5 ml LB medium containing the appropriate antibiotic, Incubate for around 8 hrs at 37C with vigorous shaking. 2. Dilute 1/500 to 1/1000 into selective LB medium. For high copy plasmids, inoculate 100 ml medium with 100-200μl of starter culture. For low copy plasmids, inoculate 250 ml medium with 250-500μl of starter culture. Grow at 37C for 12-16 hrs with vigorous shaking. 3. Spin at 6000 x g for 15 minutes at 4C 4. Resuspend pellet in 10 ml P1 Buffer 5. Add 10 ml P2 Buffer, mix thoroughly, Incubate at room temperature for 5 mins. 6. Add 10 ml chilled P3 Buffer, mix immediately and thoroughly 7. Pour into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 mins. 8. Remove the cap from the QIAfilter cartridge outlet nozzle, insert the plunger into the QIAfilter Maxi cartridge and filter the cell lysate into a 50 ml tube. 9. Add 2.5 ml ER Buffer, mix by inverting 10 times, Incubate on ice for 30 mins. 10. Equilibrate a QIAgen-Tip 500 by applying 10 ml QBT Buffer and allow the column to empty by gravity flow 11. Apply the filtered lysate from step 9 to the QIAGEN-Tip and allow it to enter the resin by gravity flow. 12. Wash the QIAGEN-Tip with 30 ml QC Buffer, Repeat QC wash 13. Elute DNA with 15 ml QN Buffer 14. Precipitate DNA by adding 10.5 ml room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥ 15,000 x g for 10 mins. Carefully decant supernatant without disturbing the pellet 15. Air-dry the pellet for 5-10 min, redissolve the DNA in a suitable volume of endotoxin-free TE Buffer.
Antibody StainingAntibody Staining Reagents: Work in an ice bucket (ethanol the bucket)
1. Pipette 1ml wash buffer into 1.5ml microcentrifuge tubes. 2. Add 1x10^5 cells into wash buffer in each tube. 3. Spin for 1 min for NKL and 2 minutes for CD8+ then aspirate. (Don’t aspirate the pellet!) 4. Go to step 3, repeat one more time. (Meanwhile, prepare FC Block) 5. Resuspend in 100ul of FC Block. Icubate in ice for 15 min. 6. Add 1ul of Goat anti-F(ab’)2 to each tube. Wrap the tube with foil. Incubate in dark for 30 minutes at 4 degrees. For the samples not being labeled, don’t add the Goat antib-F(ab’)2. 7. Add 1ml wash buffer to the Ab-labeled samples. Repeat step 3, two more times. 8. Resuspend in 250ul of wash buffer. 9. Take samples to the FACS machine. 10. Add 250ul PI solution to the samples the moment before you run the samples. TOPO cloningZero Blunt TOPO - Follow manufacturer's protocol TransfectionJurkat Cell Transfection Protocol Important: Steps 8 - 13 should be performed for each sample separately.
FACST-Cell Activation (NFAT-GFP readout by FACS) ImagingIMAGING PROTOCOL Lysotracker Blue labeling Fix the cells / permeabilize / stain with anti-GFP Alexa647 Prepare for microscopy
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