Puromycin continues to be applied for genomic purposes. In this protocol, puromycin is utilized in KO and HDR CRISPR plasmid transfections.
PHASE 1. CRISPR/Cas9 KO Plasmid and HDR Plasmid Transfection
This protocol is recommended for a single well from a 6-well tissue culture plate. Adjust cell and reagent amounts proportionately for wells or dishes of different sizes.
- In a 6-well tissue culture, plate seed 1.5x 105- 2.5x 105 cells in 3 ml of antibiotic-free standard growth medium per well 24 hours prior to transfection. Grow cells to a 40-80% confluency. Initial cell seeding and cell confluency after 24 hours are determined based on the rate of cell growth of the cells used for transfection. Healthy and subconfluent cells are required for successful KO and HDR Plasmid transfection.
- Prepare the following solutions:
NOTE: The optimal Plasmid DNA:Transfection Reagent ratio should be determined experimentally beginning with 1 µg of Plasmid DNA and between 5-15 µl of Transfection Reagent. Once the Transfection Reagent volume is optimized to minimize cell toxicity, Plasmid DNA concentrations can vary between 1-3 µg per well. If the optimal Transfection Reagent volume is 10 µl, then Plasmid DNA concentrations ranging from 1-3 µg/10 µl should be tested. For example, test Plasmid DNA/Transfection Reagent amounts 1 µg/10 µl, 2 µg/10 µl, and 3 µg/10 µl. The appropriate amount of Plasmid DNA/Transfection Reagent complex used per well should be tested to determine which amount provides the highest level of transfection efficiency.
NOTE: If transfecting more than one plasmid (i.e. CRISPR/Cas9 KO Plasmid with HDR Plasmid), mix Plasmid DNA at equivalent ratios.
Solution A: For each transfection, dilute 1-3 µg of Plasmid DNA into Plasmid Transfection Medium to bring the final volume to 150 µl. Pipette up and down to mix. Let stand for 5 minutes at room temperature.
Solution B: For each transfection, dilute 5-15 µl of Transfection Reagent with enough Plasmid Transfection Medium to bring the final volume to 150 µl. Pipette up and down to mix. Let stand for 5 minutes at room temperature.
NOTE: Do not add antibiotics to the Plasmid Transfection Medium.
- Add the Plasmid DNA solution (Solution A) dropwise directly to the dilute Transfection Reagent (Solution B) using a pipette. Vortex immediately and incubates for no less than 20 minutes at room temperature
- Prior to transfection, replace media with fresh antibiotic-free growth medium. Add the 300 µl Plasmid DNA/Transfection Reagent Complex (Solution A + Solution B) dropwise to well.
- Gently mix by swirling the plate.
- Incubate the cells for 24-72 hours under conditions normally used to culture the cells. No media replacement is necessary during the first 24 hours post-transfection. Add or replace media as needed 24-72 hours post-transfection.
- After incubation, successful transfection of CRISPR/Cas9 KO Plasmid may be visually confirmed by detection of the green fluorescent protein (GFP) via fluorescent microscopy and/or Western blot with GFP Antibody (B-2). Successful co-transfection of the CRISPR/Cas9 KO Plasmid and HDR Plasmid may be visually confirmed by detection of the red fluorescent protein (RFP) via fluorescent microscopy and/or Western blot.
- For cells transfected with CRISPR/Cas9 KO Plasmid, assay cells 48-72 hours after transfection step.
- For cells co-transfected with CRISPR/Cas9 KO Plasmid and HDR Plasmid go to Phase 2.
NOTE: If puromycin selection or Cre Vector transfection are not applicable see Phase 4.
PHASE 2. Puromycin Selection
NOTE: If cells were co-transfected with CRISPR/Cas9 KO Plasmid and HDR Plasmid, cells can be selected with media containing http://www.agscientific.com/puromycin-cell-culture-tested.htmlpuromycin.
- The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin
- 48-96 hours post-transfection, aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
- Select cells for a minimum of 3-5 days. Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
- Cells may be assayed at this point.
- For excision of the puromycin gene, proceed to Phase 3.
PHASE 3. Cre Vector Transfection
This protocol is recommended for selected cells co-transfected with CRISPR/Cas9 KO Plasmid and HDR Plasmid, and for the removal of genetic material flanked by LoxP sites.
NOTE: Follow Phase I plasmid transfection protocol for Cre Vector transfection.
PHASE 4. Cell Assay
Complete phenotypic and/or genotypic analysis may require isolation of single cell colonies to confirm complete allelic knockouts.
- For protein analysis, change media to standard growth medium 3 days prior to cell lysis. To lyse adherent cells, aspirate media, rinse cells with PBS, scrape and centrifuge cells at low speed to obtain a cell pellet. For suspension cells, transfer the culture to a centrifuge tube and centrifuge cells at low speed to obtain a cell pellet. Wash once with PBS and centrifuge again. For 100% confluent HEK 293 or HeLa cells, add 100 µl of RIPA Lysis Buffer System to the pellet. For other cell lines or confluencies, the amount of RIPA Lysis Buffer System to use should be determined experimentally. Sonicate or shear cells. Incubate sample on ice for 10 minutes, vortex, and incubate again for 10 minutes on ice. Spin cell lysate at 10000 RPM for 20 minutes at 4° C. Use the BCA Protein Assay Kitto determine protein concentration.
- For RT-PCR analysis isolate RNA using the method described by P. Chomczynski and N. Sacchi (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159) or a commercially available RNA isolation kit.
NOTE: For a listing of many reagents required to prepare the following solutions, see Core Reagents section.
- Blotto A (for general use): 1x TBS, 5% milk, 0.05% Tween-20. Available Pre-made.
- Blotto B (for use with anti-phosphotyrosine antibodies): 1x TBS, 1% milk, 1% BSA, 0.05 Tween-20. In some cases, milk may be left out entirely, but this will result in somewhat higher backgrounds. Available pre-made. For all phospho-specific antibodies: Add 0.01% (v/v) of each Phosphatase Inhibitor Cocktail to inhibit phosphatase activity.
- Diaminobenzidine tetrahydrochloride (DAB): Dissolve 5 mg DAB in 100 ml 100 mM Tris-HCl, pH 7.6, and add 0.1 ml 0.3% hydrogen peroxide. Prepare fresh DAB solution daily.
- Electrophoresis buffer (2X): 100mM 2-(N-Morpholino)- ethanesulfonic acid(MES), 10 mM Na EDTA, 15% glycerol, 1.5% SDS, 0.3% Triton X-100, 100mM TCEP-HCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Available pre-made.
- Phosphate buffered saline (1x PBS): 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, and 150 mM NaCl. Adjust pH to 7.4 with NaOH. Available pre-made in liquid and powder forms
- RIPA Lysis Buffer: 1x PBS, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS. This may be made in large volumes. Add inhibitors at time of use from the following stock solutions. Available pre-made.
1) 10 mg/ml PMSF in isopropanol (add at 10 µl/ml RIPA)
2) Aprotinin (add at 50 KIU/ml RIPA)
3) 100 mM sodium orthovanadate in frozen aliquots (add at 10 µl/ml RIPA)
- Subbing solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled H2O.
- Tris buffered saline (1x TBS): 10 mM Tris-HCl, pH 7.4; 150 mM NaCl. Available pre-made in liquid form.
PMID: 24157548, PMID: 23287718