(b) 40 ng digested and size selected pCas9counter.
(c) 2 μL T4 Ligase buffer.
(d) 1 μL T4 Ligase.
(e) Water to 20 μL.
4. Incubate at room temperature for 30 min to 1 h (up to an
overnight incubation).
5. Transform either into commercially available E. coli as per
manufacturer instructions or directly into electrocompetent
E. coli plasmid artificial modification strains as (see Subheading
3.4 and Note 3), selecting on LB media containing ampicillin
(100 μg/mL).
6. Screen 10–20 transformants for insert using colony PCR.
pCAS9counter natively contains a nonfunctional spacer region
between BsaI cut sites in the sgRNA element, which is replaced
by the shorter targeting oligonucleotides after successful
cloning.
(a) Using a sterile pipette tip, pick a transformant colony and
gently patch it to a fresh plate. Resuspend the remaining
colony in 20 μL reagent grade water, then heat to 95
for
10 min to release the DNA.
(b) Per colony screened, prepare a PCR cocktail as follows (see
Note 4):
12.5 μL KAPA HiFi Hotstart readymix (2Â).
0.75 μL sgRNA_check_F (10 μM).
0.75 μL sgRNA_check_R (10 μM).
10.35 μL water.
2 μL colony boiling preparation crude DNA extract.
(c) Amplify using the following conditions: 1 cycle of 95
for
3 min; 30 cycles of 98
for 20 s, 59
for 20 s, 72
for 30 s;
1 cycle of 72
for 1 min.
(d) Resolve PCR products by 2% agarose gel electrophoresis.
Unmodified pCAS9counter will yield a 227 bp amplicon,
whereas successful cloning products will amplify with a
133 bp band.
7. Extract plasmid DNA from a successful clone using commercially available kits. If a plasmid artificial modification strain has
not been used to directly clone the plasmid, transform plasmid
into an E. coli plasmid artificial modification strain (see Subheading 3.4) and reextract. At least 1 μg of plasmid is necessary
for optimal counterselection.
138
Kelsi Penewit and Stephen J. Salipante
(c) 2 μL T4 Ligase buffer.
(d) 1 μL T4 Ligase.
(e) Water to 20 μL.
4. Incubate at room temperature for 30 min to 1 h (up to an
overnight incubation).
5. Transform either into commercially available E. coli as per
manufacturer instructions or directly into electrocompetent
E. coli plasmid artificial modification strains as (see Subheading
3.4 and Note 3), selecting on LB media containing ampicillin
(100 μg/mL).
6. Screen 10–20 transformants for insert using colony PCR.
pCAS9counter natively contains a nonfunctional spacer region
between BsaI cut sites in the sgRNA element, which is replaced
by the shorter targeting oligonucleotides after successful
cloning.
(a) Using a sterile pipette tip, pick a transformant colony and
gently patch it to a fresh plate. Resuspend the remaining
colony in 20 μL reagent grade water, then heat to 95
for
10 min to release the DNA.
(b) Per colony screened, prepare a PCR cocktail as follows (see
Note 4):
12.5 μL KAPA HiFi Hotstart readymix (2Â).
0.75 μL sgRNA_check_F (10 μM).
0.75 μL sgRNA_check_R (10 μM).
10.35 μL water.
2 μL colony boiling preparation crude DNA extract.
(c) Amplify using the following conditions: 1 cycle of 95
for
3 min; 30 cycles of 98
for 20 s, 59
for 20 s, 72
for 30 s;
1 cycle of 72
for 1 min.
(d) Resolve PCR products by 2% agarose gel electrophoresis.
Unmodified pCAS9counter will yield a 227 bp amplicon,
whereas successful cloning products will amplify with a
133 bp band.
7. Extract plasmid DNA from a successful clone using commercially available kits. If a plasmid artificial modification strain has
not been used to directly clone the plasmid, transform plasmid
into an E. coli plasmid artificial modification strain (see Subheading 3.4) and reextract. At least 1 μg of plasmid is necessary
for optimal counterselection.
138
Kelsi Penewit and Stephen J. Salipante