TITLE: sgRNA library re-amplification in liquid culture
AUTHORS: Erik.Haussner, micboe

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: Library transformation
1. Prepare       and   for electroporation.

SECTION: Library transformation
2. Add 100 ng     into 25 µL   , carefully mix by pipetting up and down.




SECTION: Library transformation
3. 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   . 


 

 

        
SECTION: Library recovery
4. After electroporation, add the 1 mL  resuspended cells in a 14 ml culture tube and incubate the cells in a thermoshaker    .

 



SECTION: Determination of transformation efficiency
5. Use a small fraction of your cells to determine the electroporation efficiency of the reamplification. 

 


SECTION: Determination of transformation efficiency
5.1. For 1:10,000 dilution: 
Prepare   . Take  10 µL  of recovery culture and dilute in 990 µL  of   (1:100 dilution). Take 100 µL  of 1:100 dilution and dilute in 900 µL  of   (1:1,000) and plate 100 µL  on    (1:10,000 dilution).

SECTION: Determination of transformation efficiency
5.2. For 1:1,000,000 dilution: 
Take 10 µL  of the 1:1,000 dilution and dilute in 990 µL  of   and plate 100 uL on a pre-warmed   (1:1,000,000 dilution).


 


SECTION: Library extraction and quality control
7. Use rest of recovery to inoculate up to 500 mL  of    with an added selection marker specific antibiotic like ampicillin in an Erlenmeyer flask for   culture.  

SECTION: Determination of transformation efficiency
8. On the next day, check for overall coverage via colony counting on  . The overall colony count should be 1000x the element number of your library.

 

SECTION: QC: Plasmid recombination check
13. Prepare a   by melting 1 g of   in 100 mL   

SECTION: Library preparation and QC
9. Follow the protocol instructions of the    for transfection-grade plasmid DNA for Midi Prep. Follow the protocol instructions of the for transfection-grade plasmid DNA for Midi Prep.
SECTION: Determination of transformation efficiency
6. Place the plates in an incubator at 37 °C  .

SECTION: QC: Plasmid recombination check
12. 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   via a restriction digest within the backbone of your library vector. 
SECTION: QC: Plasmid recombination check
14. Let the required amount for casting cool down till it is approxametly 50 °C and add 1 µL  of   per ml of melted  
SECTION: QC: Plasmid recombination check
15. Pour the warm, still liquid gel into an electroporation chamber and wait until it has cooled down.
SECTION: QC: Plasmid recombination check
17. Add your linearized reamplified   alongside with the prepared    onto the gel and run it for 60 min at  120 V.

SECTION: QC: Plasmid recombination check
16. Mix your liearized reamplified    with    and prepare   for gel loading.

SECTION: QC: Plasmid recombination check
18. Check the plasmid size on your gel using UV excitation.

 

SECTION: Library preparation and QC
10. Determine your final    concentration via NanoDrop or Qubit measurement.
 

 


SECTION: Library preparation and QC
11. Send a sample of your reamplified   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. 
 
 
 


SECTION: Library transformation
1.1. Thaw     
5 min  on ice.
SECTION: Library transformation
1.2. Pre-cool    by placing it on ice. 
SECTION: Library transformation
1.3. Pre-warm   at 37 °C .