Protocols · Benchling

Materials

Plasmids
- Cloning: pSpCas9(BB)
- Positive control: pUC19
- Negative control: ligation of BbsI-digested pSpCas9(BB) without insert
T4 polynucleotide kinase (New England BioLabs)
- comes with T4 DNA ligase reaction buffer, 10× (New England BioLabs, cat. no. B₀₂₀₂S)
FastDigest BbsI (BpiI) (Fermentas/Thermo Scientific, cat. no. FD1014)
Fermentas Tango buffer (Fermentas/Thermo Scientific, cat. no. BY₅)
DTT (Fermentas/Thermo Scientific, cat. no. R0862)
T7 DNA ligase with 2× rapid ligation buffer (Enzymatics, cat. no. L6020L).
Adenosine 5′-triphosphate, 10mM (New England BioLabs, cat. no. P₀₇₅₆S)
PlasmidSafe ATP-dependent DNase (Epicentre, cat. no. E3101K)

One Shot Stbl3 chemically competent E. coli (Life Technologies, cat. no. C₇₃₇₃-03)
- This kit includes the competent cells, pUC19 and SOC medium
SOC Outgrowth Medium (New England BioLabs, cat. no. B₉₀₂₀S)
LB medium (Sigma, cat. no. L3022)
Ampicillin, 100mg/mL, sterile filtered (Sigma, cat. no. A5354)
QIAprep Spin Miniprep Kit
- Buffer P₁
- Buffer P₂
- Buffer N₃
- Buffer PB
- Buffer PE
- Buffer EB
- RNase A
- Collection Tubes

Step 1. Order oligos to synthesize your gRNA.
15 m

Make sure your protospacer sequence does not have BbsI enzyme site (GAAGAC or GTCTTC).

Design two oligos to synthesize your gRNA:

sgRNA-top: 5'-CACCgNNNNNNNNNNNNNNNNNNN-3'

sgRNA-bottom: 5'-AAACNNNNNNNNNNNNNNNNNNNc-3' (* the protospacer sequence here should be complementary to the sgRNA-top oligo)

Step 2. Phosphorylate and anneal each pair of the oligos
1 h

Prepare reagents for phosphorylating and annealing the gRNA oligos:

		Master mix:	2
Component	Final conc.	Amount	Master mix
gRNA top (100 uM)	10 uM	1
gRNA bottom (100 uM)	10 uM	1
10x T4 ligation buffer	1x	1	2
T4 PNK		0.5	1
ddH2O		6.5	13
Total		10

Set up reaction condition:

#	Temperature	Time
1	37 °C	30 min
2	95 °C	5 min
3	25 °C	ramp down from 95 °C with 5 °C/min

Dilute the oligos 1:250.

Step 3. Linearize the desired vector with BbsI and ligate oligos
2 h

Set up digestion-ligation reaction:

		Master mix	2
Component	Final conc	Volume (ul)	Master mix
pX330	100 ng	0.5	1
oligo duplex (diluted)		2
10X Tango Buffer	1X	2	4
DTT (10 mM)	1 mM	1	2
ATP (10 mM)	1 mM	1	2
FastDigest BbsI		1	2
T7 DNA ligase		0.5	1
ddH2O		12	24
Total		20

Set up reaction condition:

#	Temperature	Time	Action
1	37 °C	5 min
2	23 °C	5 min	Go to #1, 5 times
3	4 °C	∞

(Optional) Digest residual linearized DNA with PlasmidSafe exonuclease.

Incubate the reaction at 37°C for 30 min.

		Master mix	5
Component	Final conc	Volume (ul)	Master mix
Ligation reaction (Step 2)		11
10X PlasmidSafe buffer	1X	1.5	7.5
10 mM ATP	1 mM	1.5	7.5
PlasmidSafe exonuclease		1	5
Total		15

PAUSEAfter PlasmidSafe treatment, the reaction can be stored at -20°C for at least a week.

Step 4. Transform the final product
30 m

Before you begin:

1. Set a water bath to 42°C.

2. Warm S.O.C. medium and LB medium to room temperature.

3. Warm the selective plates to in a 37°C incubator for 30 minutes.

Thaw, on ice, one vial of 20µL Stbl3 cells for each transformation.

Add 2µL product into 20µL Stbl3 cells.

CRITICALMix gently by inverting the tube. Do NOT mix by pipetting.

Incubate the vial on ice for 10 min.

Heat-shock the cells at 42°C for 30 sec.

Immediately place the cells on ice for 2 minutes.

Add 100µL of pre-warmed SOC medium to each tube.

Optional: Incubate the tube and shake it at 37°C for 1 hour at 225 rpm.

This step is not necessary when you are transforming ampicillin-resistant plasmids. Ampicillin prevents newly divided cells from synthesizing the cell wall, which is critical for new cells' survival. However, transformed cells can synthesize ampicillin-resistant gene, the beta-lactamase enzyme, without any problems. Therefore, even if you do not incubate competent cells for the outgrowth period, you can still get transformed cells.

Plate all the medium onto an LB plate with 100µg/mL ampicillin.

Incubate it overnight at 37°C.

Step 5-1. Pick colonies
10 m

Pick 2~3 colonies from each plate to check for the correct insertion of sgRNA.

Innoculate a single colony into a 3-ml culture of LB medium with 100µg/mL ampicillin.

Incubate the culture and shake it at 37°C overnight.

Step 5-2. Isolate the plasmid from cultures by a QIAprep spin miniprep kit

(Optional) Before you start Qiagen kit for the first time:

1. Add the provided RNase A solution to Buffer P₁. Store Buffer P₁ at 2-8°C.

2. Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).

3. Check Buffers P₂ and N₃ before use for salt precipitation. Redissovlve any precipitate at 37°C.

Put 1.5mL of the incubated culture in an eppendorf.

You can either mini-prep all the culture or save some to freeze as a bacterial stock.

Spin the culture in 13,000 rpm for 1 min to pellet bacterial cells.

Resuspend pelleted bacterial cells in 250µL Buffer P₁.

Make sure there are no cell clumps.

Add 250µL Buffer P₂ and mix by inverting the tube 4-6 times.

This step is to lyse the cell. Do not allow the reaction to proceed for more than 5 min.

Mix it until the solution becoms viscous and slightly clear.

CRITICALDo not vortex. It will shear the genomic DNA.

Add 350µL Buffer N₃ and mix by inverting the tube 4-6 times.

Immediately after adding Buffer N₃, the solution should become cloudy.

This step is for precipitation. Mix the solution gently but thoroughly to avoid localized precipitation.

Spin for 10 min at 13,000rpm.

Apply the supernatants to the QIAprep spin column by decanting or pipetting.

Spin for 30~60 sec.

Discard the flow-through.

(Optional): Wash the QIAprep spin column by adding 500µL Buffer PB. Spin for 30-60 sec and discard the flow-through.

This step is to remove trace nuclease activity. It is necessary for strains with high levels of nuclease activity (endA+ strains) or high carbohydrate content, such as HB₁₀₁ and its derivatives.

Wash QIAprep spin column by adding 750µL Buffer PE.

Spin for 30-60 sec.

Discard the flow-through.

Spin for additional 1 min to remove residual wash buffer.

CRITICALIt is important to remove residual wash buffer. Residual ethanol from Buffer PE might affect your downstream applications.

Place the QIAprep column in a clean 1.5mL microcentrifuge tube.

Add 30~50µL Buffer EB or water for elution.

To recover maximal amount, let Buffer EB or water stand for 1 min.

Spin 1 min and the DNA will be in the flow-through.

Use nanodrop or other instruments to measure the concentration and purity of the DNA.

Step 5-3. Validate the sequence of each gRNA plasmid

Verify the sequence of each company by sequencing from the U₆ promoter using the U₆-Fwd primer.

Use Cbh-Fwd and SXRP002-007 primers to sequence the Cas9 genes. You can see the information of the primers on pX330.

Materials

Step 1. Order oligos to synthesize your gRNA. 15 m

Step 2. Phosphorylate and anneal each pair of the oligos 1 h

Step 3. Linearize the desired vector with BbsI and ligate oligos 2 h

Step 4. Transform the final product 30 m