TITLE: Rapid and robust cloning of sgRNA expression plasmids
AUTHORS: ghanem.elkassem, micboe

[DESCRIPTION]
In this lab protocol, we will outline a step-by-step procedure for cloning of spCas9 sgRNAs using oligo annealing and T4 ligation. This protocol will guide you through the crucial steps of designing, annealing, ligating oligonucleotides and digested plasmids, as well as heat shock transformation procedure to clone sgRNAs expression vectors.

[BEFORE_START]
Modify the ssOligo overhangs and Sanger sequencing primer according to the plasmid that you are using.

[STEPS]
SECTION: Oligo Design
1. Order TOP and BOTTOM strand oligos, where the TOP sequence is the desired 20 nt sgRNA 'spacer' sequence and the BOTTOM sequence is the reverse complement of the TOP strand. After annealing of both oligos, the 5' overhangs should result in compatible sticky ends for T4 ligation into the target vector. Shown are example sticky ends to clone into the vector sgLenti (Addgene #105996). Alternative sgRNA expression vectors may require different sticky ends.

5’-TTGGNNNNNNNNNNNNNNNNNNN -3’
                    3’-NNNNNNNNNNNNNNNNNNNCAAA-5’

TOP strand oligo: 5'-TTGGNNNNNNNNNNNNNNNNNNN-3'
BOTTOM strand oligo: 5'-AAACNNNNNNNNNNNNNNNNNNN-3'

SECTION: Oligo Annealing
2. Set up the annealing reaction:
1 µL Top strand oligo 100 micromolar (µM)
1 µL Bottom strand oligo 100 micromolar (µM) 
1 µL  
7 µL ddH2O
SECTION: Plasmid Restriction Digest
4. Set up AarI plasmid digest:
1 µg vector for spCas9 sgRNA expression
2 µL 10x AarI Buffer
1 µL   
0.4 µL 50x Oligo
ddH2O to 20 µL
SECTION: Plasmid Restriction Digest
5. Incubate in a Thermocycler at 50 °C   
SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
7. Add 4 µL of 6x   to 20 µL of the restriction digestion mix.

SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
8. Load samples on the 1 % Agarose gel and run the electrophoresis at 120 V for 50 min

SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
10. Cut the digested plasmid band from the gel and purify the DNA using the   

SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
11. Determine the concentration of the purified plasmid DNA (e.g. NanoDrop One)
SECTION: Ligation of Plasmid and Annealed Oligos
12. Dilute the annealed oligos 1:200 in ddH2O
SECTION: Ligation of Plasmid and Annealed Oligos
13. Set up the ligation reaction:
50 ng purified vector
1 µL diluted oligos
1 µL 10x T4 Ligation buffer
1 µL   
ddH2O to 10 µL

 


SECTION: Ligation of Plasmid and Annealed Oligos
14. Incubate for 10 min at Room temperature 
SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
15. Take DH5a chemocompetent cells from -80 °C storage and thaw on ice for 30 min. Use 1 50 µL aliquot for the ligation reaction and 1 50 µL aliquot for the control reaction
SECTION: Plasmid DNA Pr1dification
24. Pick up to 3 colonies from the plate using a pipette tip and inoculate 5 mL LB medium with 100 µg/mL Carbenicillin in 15 mL cell culture tubes


SECTION: Plasmid DNA Pr1dification
25. Incubate the cell culture tubes   at 37 °C and 225 rpm in a shaking incubator

SECTION: Plasmid DNA Pr1dification
26. Extract the plasmid DNA using the   

SECTION: Plasmid DNA Pr1dification
27. Measure the concentration of the purified plasmid DNA (e.g. NanoDrop One)
SECTION: Plasmid DNA Pr1dification
28. Send the plasmids for Sanger sequencing with the following primer to confirm the correct sequence:
GGCTTGGATTTCTATAACTTCGTATAGCA

 

SECTION: Oligo Annealing
3. Anneal oligos by running the following thermocycler program:
1. 95 °C for 5 min
2. Ramp down at 0.1 °C from 95 °C  to 25 °C 
SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
6. Prepare 1 % Agarose gel

SECTION: Digested Plasmid Purification by Agarose Gel Electrophoresis
9. Place the gel in a UV light box

 

SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
16. Add 2 µL of the T4 Ligation reaction and the control reaction to the cells and mix gently by flicking the tube
SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
17.  Incubate on ice for 30 min
SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
18. Incubate the tube containing the cells in a water bath at 42 °C for 45 sec and place back on ice for 2 min

SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
19. Add 0.5 mL of LB medium to the cells and incubate in a thermo-block at 37 °C with shaking at 250 rpm for 1 hr

SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
20. 
Plate 100 µL of the cells on Room temperature LB agar plates with 100 µg/mL Carbenicillin
SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
21. Incubate at 37 °C  

SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
22. After the overnight incubation, count the colonies on the ligation reaction and the control plates. The ligation reaction plate should have a minimum of 10x more colonies.
SECTION: Heat Shock Transformation of the Ligation Product into Chemically Competent E. Coli
23. Seal the plates with parafilm and store at 4 °C for up to 2 weeks