In vitro transcription of guide RNAs
No PCR cleanup necessary at this point .
Incubate transcription mix for ~ 18 hours at 37˚ in a thermalcycler .
Add 1 µl of RNase - free DNase ; incubated 20 min , room Temp .
Bring volume to 150 uL with 100 % EtOH ( this helps binding of small fragments ) .
Add 5X SPRI ( we use homemade SeraPure beads for RNA binding ) 5 * 10 ( IVT sgRNA ) = 50 uL of SPRI Beads5 * 20 ( IVT sgRNA ) = 100 uL SPRI Beads .
Pipette to mix 10 times Incubate 5 minutes at room temperature .
Place on magnetic stand , 5 min .
Discard supernatant .
Wash # 1 Add 200 uL , 80 % EtOH .
Wait 2 min .
Remove EtOH .
Wash # 2 : Add 200 uL , 80 % EtOH .
Wait 2 min .
Remove EtOH .
Air dry 5 - 10 min ( pellet will change from a glossy / wet to matte / dry . )
Elute 20 uL of water or TE .
Pipette mix 10 times .
Incubate 2 minutes at room temperature .
Place on magnetic stand , 5 min .
Keep Supernatant .
Transfer to a new plate / tubes .
