Oct 29, 2023
Version 1
sgRNA library re-amplification in liquid culture V.1
- 1Medical Faculty of the Martin Luther University Halle-Wittenberg
Protocol Citation: Erik Haussner, Michael Böttcher 2023. sgRNA library re-amplification in liquid culture. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmr7xl5b/v1
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: September 15, 2023
Last Modified: October 29, 2023
Protocol Integer ID: 87835
Keywords: sgRNA, reamplification, Plasmid pool, Library, Electroporation, amplification
Abstract
In this protocol, we describe a stepwise procedure for the re-amplification of sgRNA libraries in liquid culture. In our hands, this protocol works reliably to amplify pre-cloned sgRNA libraries (e.g. order from Addgene) in a way that preserves the distribution of library elements.
Materials
- 100 µL ElectroMAX™ Stbl4™ Competent CellsThermo FisherCatalog #11635018
- Up to 400 ng of Sample
- Electroporator and Electroporation cuvettesBiozymCatalog #748010
Protocol materials
ElectroMAX™ Stbl4™ Competent CellsThermo FisherCatalog #11635018
In Materials and 3 steps
TriTrack DNA Loading Dye (6X)Thermo FisherCatalog #R1161
Step 16
1 kb Plus DNA-LadderThermo Fisher ScientificCatalog #10787018
In 2 steps
1.5 mL Eppendorf tubes
Step 5.1
NucleoBond Xtra Midi kit for transfection-grade plasmid DNAMacherey-NagalCatalog #REF 740410.50
Step 9
1% Agarose gelCatalog #/
In 2 steps
Electroporation cuvettesBiozymCatalog #748010
In Materials and 2 steps
1x TBE buffer
Step 13
GelRed™ Nucleic Acid Gel Stain, 10,000X in WaterGold BiotechnologyCatalog #G-725
Step 14
SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S
In 3 steps
Liquid LB medium
In 4 steps
LB agar plates with the proper antibiotic (e.g. Kanamycin)
In 3 steps
Agarose Low MeltCarl RothCatalog #6351.4
Step 13
Before start
Keep your original stock safe and aliquoted. For large plasmids with complementary sequences such as LTR sites, keep in mind that repeated reamplification from an already reamplified stock may lead to an accumulation of recombined plasmids and a poorer distribution of library elements.
Library transformation
Library transformation
25m
Prepare Sample ElectroMAX™ Stbl4™ Competent CellsThermo FisherCatalog #11635018 Electroporation cuvettesBiozymCatalog #748010 and SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S for electroporation.
5m
Thaw ElectroMAX™ Stbl4™ Competent CellsThermo FisherCatalog #11635018
00:05:00 on ice.
5m
Pre-cool Electroporation cuvettesBiozymCatalog #748010 by placing it on ice.
Pre-warm SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S at 37 °C .
Add 100 ng Sample into 25 µL ElectroMAX™ Stbl4™ Competent CellsThermo FisherCatalog #11635018 , carefully mix by pipetting up and down.
10m
Add 25 µL of the plasmid/cell mix into a cuvette, electroporate at 1.2 kV, 25 uF and 200 ohm or alternative setting (see note below). Directly after electroporation, add 1 mL of pre-warmed SOC Outgrowth Medium - 100 mlNew England BiolabsCatalog #B9020S .
Note
The electroporator setting may vary from model to model and should be checked along with the test plasmids provided in the kit of the STBL4 cells.
Safety information
Make sure that any water or ice residue is removed from the cuvettes before inserting them into the electroporator to avoid arching.
10m
Library recovery
Library recovery
1h 15m
After electroporation, add the 1 mL resuspended cells in a 14 ml culture tube and incubate the cells in a thermoshaker 600 rpm, 37°C, 01:00:00 .
Note
In general, an incubation temperature of 37°C is optimal for cell recovery. Since E. coli tend to recombine plasmids with complementary sequences (e.g. LTRs), recovery temperature can be reduced to 30°C. This may however, result in a lower total number of recovered cells.
1h 15m
Determination of transformation efficiency
Determination of transformation efficiency
16h 30m
Use a small fraction of your cells to determine the electroporation efficiency of the reamplification.
Note
In this step, much depends on the size of the particular plasmid and the number of elements in the library. Therefore, the dilution factor must be chosen based on properties of the library and the scale of the electroporation. Smaller plasmids yield significantly more colonies than large ones, and an upscaled plasmid input at the electroporation step may result in higher dilutions being required to achieve a countable number on the respective agar plates after plating.
For 1:10,000 dilution:
Prepare 1.5 mL Eppendorf tubesContributed by users . Take 10 µL of recovery culture and dilute in 990 µL of Liquid LB mediumContributed by users (1:100 dilution). Take 100 µL of 1:100 dilution and dilute in 900 µL of Liquid LB mediumContributed by users (1:1,000) and plate 100 µL on LB agar plates with the proper antibiotic (e.g. Kanamycin)Contributed by users (1:10,000 dilution).
5m
For 1:1,000,000 dilution:
Take 10 µL of the 1:1,000 dilution and dilute in 990 µL ofLiquid LB mediumContributed by users and plate 100 uL on a pre-warmed LB agar plates with the proper antibiotic (e.g. Kanamycin)Contributed by users (1:1,000,000 dilution).
Note
When preparing the dilution series, always mix stock solutions well by flicking the tube before diluting, to resuspend sedimented cells. Distribute the plated cells evenly over the plate by e.g. using glas beads.
5m
Place the plates in an incubator at 37 °C Overnight .
16h
Library extraction and quality control
Library extraction and quality control
16h
Use rest of recovery to inoculate up to 500 mL of Liquid LB mediumContributed by users with an added selection marker specific antibiotic like ampicillin in an Erlenmeyer flask for Overnight culture.600 rpm, 30°C
16h
Determination of transformation efficiency
Determination of transformation efficiency
15m
On the next day, check for overall coverage via colony counting on LB agar plates with the proper antibiotic (e.g. Kanamycin)Contributed by users . The overall colony count should be 1000x the element number of your library.
Note
Below we provide a simplified example for how to determine transformation coverage.
Example calculation of coverage: On the 1:10,000 dilution plate we count 100 colonies. This gives us 100*10,000 = 1,000,000 total colonies. This total colony number is divided by the number of elements (e.g. sgRNAs) in the respective library. For a library the size of 1,000 sgRNAs, the coverage would 1,000,000/1,000=1,000x. For larger libraries, e.g. the size of the genome-wide Brunello library (80,000 sgRNAs), we would count the 1:1,000,000 dilution plate. In this case, 80 counted colonies would mean 80,000,000 total colonies which divided by the library size (80,000 sgRNAs) would again return a transformation coverage of 1,000x.
15m
Library preparation and QC
Library preparation and QC
1h
Follow the protocol instructions of the NucleoBond Xtra Midi kit for transfection-grade plasmid DNAMacherey-NagalCatalog #REF 740410.50 for transfection-grade plasmid DNA for Midi Prep. Follow the protocol instructions of the for transfection-grade plasmid DNA for Midi Prep.
25m
Determine your final Sample concentration via NanoDrop or Qubit measurement.
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
Send a sample of your reamplified Sample for Sanger sequencing.
Below we show an example chromatogram of an expected sequencing result. We recommend using sequencing primers 50-100 nt upstream of the sgRNA region. You should see clean traces up- and downstream of the SPACER region, and a noisy 20 nt signal in the SPACER region, due to the sgRNA diversity in your library.
Example of a Sanger sequencing result.
Note
! To validate the distribution of elements in your library, we strongly recommend performing next generation sequencing of your plasmid pool before proceeding with downstream experiments. To do so, follow the NGS protocol provided with your library, using the plasmid pool as template, instead of the genomic DNA (as you would in CRISPR screens).
QC: Plasmid recombination check
QC: Plasmid recombination check
3h 15m
Since sgRNA library plasmids can recombine during E.coli re-amplification, it is recommended to check for recombination via linearization of 200 ng of your reamplified Sample via a restriction digest within the backbone of your library vector.
1h
Prepare a 1% Agarose gelContributed by usersCatalog #/ by melting 1 g of Agarose Low MeltCarl RothCatalog #6351.4 in 100 mL 1x TBE bufferContributed by users
20m
Let the required amount for casting cool down till it is approxametly 50 °C and add 1 µL of GelRed™ Nucleic Acid Gel Stain, 10,000X in WaterGold BiotechnologyCatalog #G-725 per ml of melted 1% Agarose gelContributed by usersCatalog #/
30m
Pour the warm, still liquid gel into an electroporation chamber and wait until it has cooled down.
15m
Mix your liearized reamplified Sample with TriTrack DNA Loading Dye (6X)Thermo FisherCatalog #R1161 and prepare 1 kb Plus DNA-LadderThermo Fisher ScientificCatalog #10787018 for gel loading.
5m
Add your linearized reamplified Sample alongside with the prepared 1 kb Plus DNA-LadderThermo Fisher ScientificCatalog #10787018 onto the gel and run it for 01:00:00 at 120 V.
1h
Check the plasmid size on your gel using UV excitation.
Expected result
In the best case, only one band will be present, corresponding to the size of the respective vector. In case of recombination, one or more additional bands will be present. For optimal downstream results, the band of the intact vector should be dominant.
5m