rpoB_rv0667 _T: 5
0 -GGGAGACATCGTCGAAACGAGGGTC-3
0 .
rpoB_rv0667_B: 5
0 -AAACGACCCTCGTTTCGACGATGTC-3
0 .
mmpL3_ms0250_T: 5
0 -GGGAGCGACAGACTGGCTGCCCT
CGTC-3
0 .
mmpL3_ms0250_B: 5
0 -AAACGACGAGGGCAGCCAGTCTGT
CGC-3
0 .
3.2 Annealing sgRNA
Oligos for Ligation into
the CRISPRi Plasmid
Backbone
The oligos can be annealed by heating the oligo mixture to 95
C,
then slowly reducing the solution to ambient temperature to allow
annealing of the oligos.
1. Set up the oligo annealing reaction in PCR tubes as below:
46 μL oligo annealing buffer.
2 μL 100 μM top oligo.
2 μL 100 μM bottom oligo.
2. Place the PCR tubes in a thermocycler and anneal the oligos
using the following cycling conditions:
95
C for 2 min.
À0.1
C/s to 25
C.
END.
3. Dilute the annealed oligos 1:100 in deionized water.
3.3 BsmBI-Digestion
of the CRISPRi Plasmid
Backbone
1. Set up a BsmBI restriction digest as follows:
5 μL NEBuffer 3.1.
4 μL BsmBI-v2 (10,000 U/mL).
2–5 μg CRISPRi backbone (plJR965).
Water up to 50 μL.
2. Incubate at 55
C for 4 h—overnight.
3. Cast a 0.7% (w/v) TAE agarose gel with a blue-light transilluminator compatible, DNA intercalating dye (such as
SYBR Safe).
4. Load the entire digestion reaction on the gel. As a control to
assess efficient plasmid digestion, also load 100 ng of undigested plJR965, leaving at least one empty lane between the
control and the digestion reaction.
5. Run the DNA gel at 120 V for approximately 45 min.
6. Use a blue-light transilluminator to visually confirm BsmBI
digestion of the CRISPRi plasmid. The digested, linear plasmid
should be approximately 8.6 kb and migrate at a higher molecular weight than the undigested, supercoiled plasmid.
7. Gel extract the digested CRISPRi backbone using the QIAquick Gel Extraction Kit.
352
Andrew I. Wong and Jeremy M. Rock
0 -GGGAGACATCGTCGAAACGAGGGTC-3
0 .
rpoB_rv0667_B: 5
0 -AAACGACCCTCGTTTCGACGATGTC-3
0 .
mmpL3_ms0250_T: 5
0 -GGGAGCGACAGACTGGCTGCCCT
CGTC-3
0 .
mmpL3_ms0250_B: 5
0 -AAACGACGAGGGCAGCCAGTCTGT
CGC-3
0 .
3.2 Annealing sgRNA
Oligos for Ligation into
the CRISPRi Plasmid
Backbone
The oligos can be annealed by heating the oligo mixture to 95
C,
then slowly reducing the solution to ambient temperature to allow
annealing of the oligos.
1. Set up the oligo annealing reaction in PCR tubes as below:
46 μL oligo annealing buffer.
2 μL 100 μM top oligo.
2 μL 100 μM bottom oligo.
2. Place the PCR tubes in a thermocycler and anneal the oligos
using the following cycling conditions:
95
C for 2 min.
À0.1
C/s to 25
C.
END.
3. Dilute the annealed oligos 1:100 in deionized water.
3.3 BsmBI-Digestion
of the CRISPRi Plasmid
Backbone
1. Set up a BsmBI restriction digest as follows:
5 μL NEBuffer 3.1.
4 μL BsmBI-v2 (10,000 U/mL).
2–5 μg CRISPRi backbone (plJR965).
Water up to 50 μL.
2. Incubate at 55
C for 4 h—overnight.
3. Cast a 0.7% (w/v) TAE agarose gel with a blue-light transilluminator compatible, DNA intercalating dye (such as
SYBR Safe).
4. Load the entire digestion reaction on the gel. As a control to
assess efficient plasmid digestion, also load 100 ng of undigested plJR965, leaving at least one empty lane between the
control and the digestion reaction.
5. Run the DNA gel at 120 V for approximately 45 min.
6. Use a blue-light transilluminator to visually confirm BsmBI
digestion of the CRISPRi plasmid. The digested, linear plasmid
should be approximately 8.6 kb and migrate at a higher molecular weight than the undigested, supercoiled plasmid.
7. Gel extract the digested CRISPRi backbone using the QIAquick Gel Extraction Kit.
352
Andrew I. Wong and Jeremy M. Rock