Overview

This workflow describes the steps to follow immediately after performing a CRISPR knockout. The goal is to expand the edited cells, quantify them, extract RNA for cDNA and qPCR, and confirm protein loss with a Western blot using a low-cost ECL plus smartphone imaging method. This SOP references the Counting Cells (Post-Experiment), RNA Purification, RT-PCR (cDNA), and Realtime (qPCR) protocols.

Materials

CRISPR edited cells
Positive control cells
Negative control cells
Standard culture media
Hemocytometer or automated counter
RNA extraction kit (RNA Purification SOP)
cDNA synthesis reagents (RT-PCR SOP)
qPCR reagents and primers (Realtime qPCR SOP)
RIPA or other protein lysis buffer
ECL substrate
Plastic sheet protector
Black background surface
Smartphone with manual exposure controls
Labeled cryovials

Step 1. Cell Recovery and Expansion (Day 0 to Day 3)

Allow CRISPR edited cells to recover and grow for 48 to 72 hours. Monitor morphology and viability. Do not aggressively passage unless overcrowded. Goal is to reach at least 1 to 2 million total cells.

Control Sample Processing (Positive and Negative Controls)

Positive and negative control samples should be processed immediately and frozen for later validation. The knockout and matched wild type cells must remain growing.

Transfer each control culture into a labeled 1.5 mL microtube or 15 mL conical tube (depending on the well volume used).
Centrifuge at 300 g for 5 minutes.
For a clinical centrifuge (15 mL tubes): use ≈ 1,500 rpm.
For a microcentrifuge (1.5 mL tubes): use ≈ 4,000 rpm.
Decant the supernatant without disturbing the pellet.
Freeze at minus 20°C.

These controls serve as reference material. Only the knockout and wild type cells (control) should remain in active culture.

Step 2. Cell Counting (Use the Counting Cells Post-Experiment SOP)

Count the knockout and wild type cells using Trypan Blue viability. Record total cell number and viability percentage.

Allocate cells as follows:

300k to 1 million cells for RNA extraction
500k to 1 million cells for protein lysate
Remaining cells should be expanded in a fresh flask for continued growth

Step 2.5. Preparing Cells for RNA or Protein Collection

Use this procedure to collect experimental wells and control wells while keeping 30 to 50 percent of edited cells growing for future work.

Save 30 to 50 percent of cells for future experiments.
Work aseptically in the cell culture hood.
Label one 1.5 mL microtube or 15 mL conical tube (depending on the well volume used) for each well.
Only process experimental, positive, and negative wells.
Aspirate supernatant carefully using separate Pasteur pipettes.
Add 1 mL TE to each well and incubate at 37°C for 5 minutes.
Rinse wells to detach cells using new tips for each sample.
Check detachment under the microscope.
Transfer suspended cells to conical tubes. For positive and negative controls, transfer everything. For experimental wells, place 500 µL into a new flask with 7 mL medium to keep the CRISPR edited culture alive. Place the remaining 500 µL into the conical tube.
Add 1 mL fresh medium to each conical tube to dilute TE.
Spin at 2000 g for 5 minutes at 4°C.
Aspirate supernatant and proceed to lysis or freeze the pellet at minus 80°C.

Step 3. RNA Extraction (Use the RNA Purification SOP)

Extract total RNA from the collected cell pellets using the RNA Purification SOP.

RNA from 300k to 1 million cells is sufficient for all downstream applications.

Step 4. Generate cDNA (Use the RT-PCR SOP)

Synthesize cDNA from purified RNA using the RT-PCR (cDNA) protocol.

Ensure enough cDNA is prepared for multiple qPCR replicates.

Step 5. Confirm Transcript Loss by qPCR (Use the Realtime qPCR SOP)

Perform qPCR to quantify gene expression levels in knockout versus wild type cells.

Interpretation:

Strong reduction or absence of transcript indicates likely knockout
Partial reduction may indicate mixed populations

Step 6. Protein Lysate Preparation

Prepare protein lysates from 0.5 to 1 million cells using RIPA or another lysis buffer. Quantify protein and normalize before loading the gel.

Step 7. Western Blot and Imaging (ECL plus Smartphone)

Blotting

Perform SDS-PAGE, transfer proteins to a membrane, and complete antibody incubations as usual.

Imaging with ECL and Smartphone

Place the membrane in a sheet protector, add ECL substrate, and image using a smartphone in a dark room.

Lay the membrane on a black background.
Turn off room lights.
Use a manual camera app with adjustable ISO and shutter speed.
Suggested settings: ISO 400 to 1600, shutter 2 to 10 seconds.
Capture several images and save the highest resolution version.

This method replaces X-ray film and developer and provides strong chemiluminescent signal for knockout confirmation.

Step 8. Data Interpretation

qPCR: Confirms transcript loss or reduction
Western blot: Confirms protein loss

Together these assays validate the CRISPR knockout at transcript and protein levels.

Next Steps (Optional)

Single cell cloning to isolate pure knockout lines
Functional assays such as proliferation or apoptosis
Freezing a master stock of knockout cells