Protocols_MicroBiology_gRNA cloning protocol updated - BackmanLab/BackmanLab.github.io GitHub Wiki

gRNA cloning (3 days)

Step 0: Spin down the lyophilized primer tubes at 13000 rpm for a minute. Reconstitute the gRNA primers with ddH20/DEPC-water/mol bio water to 100 uM (For ex. 223 ul in 22.3 nmol primer, nmol info is provided on the primer tube label). Proceed to annealing (for generating double stranded gRNA) and phosphorylation (for ligation to gRNA cloning backbone).

  1. Primer Annealing and phosphorylation

sgRNA FP (1oo μM) 1 μL

sgRNA RP (1oo μM 1 μL T4 ligation buffer 10X 1 μL T4 PNK 1 μL ddH2O 6 μL Total volume 10 μL

37 °C for 30 min, 95 °C for 5 m, ramp down to 25 °C for 6 °C min^-1^

(I have an annealing thermocycling program set up in the thermocycler in the folder "Surbhi")

  1. Above mixture is diluted 200X with ddH2O. (1 ul of annealed primer in 199 ul of ddH2O)

Ligated to gRNA backbone (empty vector)

 

Backbone 100 ng (x ul)

Diluted oligo duplex 2 μL NEB Cutsmart 10X 2 μL T4 DNA ligase 1 μL DTT 10 mM 1 μL ATP 10 mM 1 μL BbsI 1 μL Water (16-x) ul Total volume 20 μL

 

  1. cycles of 37 °C for 5 min and 16 °C for 10 min.
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  1. Treat the ligation reaction with PlasmidSafe exonuclease to digest any residual linearized DNA. This step is optional but highly recommended.

Component Amount (μl)

Ligation reaction from Step 2 11 PlasmidSafe buffer, 10× 1.5 ATP, 10 mM 1.5 PlasmidSafe exonuclease 1 Total 15

Incubate the PlasmidSafe reaction at 37 °C for 30 min, followed by 70 °C for 30 min.

PAUSE POINT

After PlasmidSafe treatment, the reaction can be stored at −20 °C for at least 1 week.

  1. Transformation using Stellar cells, and plate on Amp resistant plate.
    (Protocol source: https://www.takarabio.com/documents/User%20Manual/PT5055-2.pdf)

    a. Thaw Stellar Competent Cells in an ice bath just before use.

    b. After thawing, mix gently to ensure even distribution, and then move 50 μl of competent cells into a 14-ml round bottom tube (falcon tube). Do not vortex.

    c. Add no more than 5 ng of DNA for transformation.

    d. Place tubes on ice for 30 min.

    e. Heat shock the cells for exactly 45 sec at 42°C.

    f. Place tubes on ice for 1--2 min.

    g. Add SOC medium to bring the final volume to 1ml. SOC medium should be warmed to 37°C before using.

    h. Incubate by shaking (160--225 rpm) for 1 hr at 37°C.

    i. Plate an appropriate amount of culture on selective medium.

    j. Incubate overnight at 37°C.

NOTE: For a plate with a diameter of 9 cm, plate 100 μl. Plating is accomplished by spreading cells on selective medium [e.g., LB agar + Ampicillin (50--100 μg/ml)]. The medium should also contain X-Gal (40 μg/ml) for plasmids that permit blue/white screening of transformants.

  1. The next day, pick 1 colony into 5ml culture with Amp or antibiotic appropriate for your plasmid, shake at 210-220 rpm at 37C overnight.

  2. Miniprep the culture and send for sequencing with your sequencing primer.

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