Jan 16, 2018
Cas9/sgRNA ribonucleoprotein nucleofection using Lonza 4D nucleofector
- 1University of California, San Francisco
- Stephen Floor Lab
Protocol Citation: Bao Thai 2018. Cas9/sgRNA ribonucleoprotein nucleofection using Lonza 4D nucleofector. protocols.io https://dx.doi.org/10.17504/protocols.io.mpfc5jn
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 9671
Keywords: sgrna ribonucleoprotein nucleofection, using lonza 4d nucleofector, lonza 4d nucleofector, cas9
Materials
STEP MATERIALS
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
Amaxa SF Cell Line 4D-Nucleofector Kit S (96 RCT)LonzaCatalog #V4SC-2096
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
Amaxa SF Cell Line 4D-Nucleofector Kit S (96 RCT)LonzaCatalog #V4SC-2096
Protocol materials
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
Amaxa SF Cell Line 4D-Nucleofector Kit S (96 RCT)LonzaCatalog #V4SC-2096
Prepare cells (part 1)
Trypsinize cells and spin down at 100 x g for 5 minutes.
Remove trypsin and resuspend cells in an appropriate amount of fresh media.
Count cells. Record the cell concentration (cells/uL). In the meantime, put media containing cells in a 37C water bath.
Prepare ribonucleoproteins (RNPs) mix
Add 2.5 uL of 40mM Cas9 (~100 pmol) to 2.5 uL of Cas9 buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% glycerol, 1 mM TCEP-can make this ahead of time, aliquot and store at -20C).
Add 3880 ng of sgRNA (~120 pmol, MW~32,327g/mol) to Cas9 buffer totaling 5 uL.
Add Cas9 to sgRNA slowly while swirling pipette tip.
Incubate at 37C for 10-20 minutes to let RNP form.
Prepare cells (part 2)
For each nucleofection, pipette 200k cells using a P200 or larger into a 1.5 mL tube.
Spin 100 x g for 10 minutes to pellet cells softly.
While the cells are spinning, prepare a 12-well plate containing 1 mL of media per well. Pre-warm at 37C.
Nucleofection
Prepare and label wells on nucleofection cuvettes. To avoid cells staying in nucleofection solution for a long period of time in the subsequent steps, configure Lonza 4D ahead of time using the recommended cell-type program. Use SF cell line program CM-130 for HEK293T cells.
Lonza Nucleofector 4dLonzaCatalog #AAF-1002X
Amaxa SF Cell Line 4D-Nucleofector Kit S (96 RCT)LonzaCatalog #V4SC-2096
After centrifugation, cell pellets are soft so carefully remove media from cells.
Resuspend cells in 20 uL of nucleofector solution (SF cell line solution with added supplement for HEK293T) using a P200.
Add the entire 10 uL RNP mix to the 20 µL resuspension and mix using a P200.
If using a repair template, add 1uL of 100uM single-stranded donor DNA (100 pmoles) and mix well.
Add nucleofection mixes to the multiwell cuvette, and cap.
Insert cuvette into nucleofector and zap using the configured program.
Allow cells to sit in nucleofection strips for 10 minutes post-nucleofection. This is supposed to increase efficiency.
Add 80uL of pre-warmed media to each well. Pipette mixture out with a P200 into your pre-warmed 12-well plate.
Allow cells 24 hours to settle and recover before attempted downstream analysis. Consider including un-zapped controls to test viability.