5. Incubate the microfuge tube in the shaker at 37
C for 1 h at
250 rpm.
6. Centrifuge at 2000 Â g for 5 min.
7. Aspirate 500 μL of the LB medium, resuspend the cell pellet,
and spread 150 μL of cell suspension onto an LB agar plate
containing 100 μg/mL ampicillin.
8. Incubate at 37
C overnight (see Note 7).
9. Pick up 3–5 single colonies, inoculate 6 mL of LB medium
supplemented with 100 μg/mL ampicillin, incubate in the
shaker at 37
C at 250 rpm overnight.
10. Aliquot 5 mL of culture for plasmid extraction using Presto™
Mini Plasmid Kit, following the manufacturer’s instruction and
store 1 mL of culture at 4
C for short-term storage. Measure
the concentration and quality of the sgRNA/PX459 plasmid
using NanoDrop 2000. Store the plasmid at À20
C.
3.3.3 Sequence
Validation of CRISPR
Plasmid
At Least 100 Ng of the Recombinant plasmid Is Required
for Sequencing.
1. Verify the sequence of the isolated sgRNA/PX459 plasmid by
sequencing from the U6 promotor using the U6-Forward
primer (Table 2).
2. Select the bacterial culture with the correct DNA sequence for
plasmid preparation using Geneaid™ Midi Plasmid Kit for the
nucleofection step. Add 1 mL of culture that has been stored at
4
C (from Subheading 3.3.2, step 10) into fresh 100 mL of
LB medium containing 100 μg/mL ampicillin and incubate in
the shaker at 37
C at 250 rpm overnight.
Table 6
Components for ligation
Components
Amount (μL) for 1 reaction
PX459, 100 ng
X
Diluted oligo duplex (Subheading 3.2, step 6)
2
Tango buffer, 10Â
2
DTT, 10 mM
1
ATP, 10 mM
1
FastDigest BbsI
1
T7 ligase
0.5
ddH 2 O
U p t o 2 0
Total
20
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