5. Purify the PCR product by using the GenepHlow™ gel/PCR
kit according to the manufacturer’s instruction and further
confirm the result with Sanger sequencing.
6. Analyze the sequencing data using the read alignment programs such as MUSCLE (https://www.ebi.ac.uk/Tools/
msa/muscle/).
4 Notes
1. The length of homology arms of the ssODN repair template
can range from 90–180 nucleotides. The ssODN repair template should be HPLC purified.
2. Thaw the mTeSR™1 5Â supplement at room temperature or 4
C for overnight. Do not thaw the supplement at 37
C.
Prepare complete mTeSR™1 medium by mixing the
mTeSR™1 5Â supplement and the mTeSR™1 basal medium.
Store the complete mTeSR™1 medium at 2–8
C for up to
Table 9
Reaction setup for off-target analysis
Component
Amount (μL)
Final concentration
Q5 high-Fidelity DNA polymerase, 100Â
0.5
0.2 U
Q5 reaction buffer with MgCl 2 , 5Â
10
1Â
dNTPs, 10 mM
1
0.2 mM
Each primer, 10 μM
2.5
0.5 μM
DNA sample
2
Nuclease-free water
To 50
Table 10
Thermal cycling conditions for PCR verification of clones
Step
Temperature and time
Initial denaturation
98
C, 30 s
35 cycles
98
C, 10 s
68
C for 30 s
72
C for 30 sec
Final extension
72
C for 2 min
Hold
4
C
208
Methichit Wattanapanitch