TITLE: CasX GFP-Targeting gRNA IVT
AUTHORS: Connor Tsuchida, Liz O'Brien

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to transcribe gRNA in vitro. </div></div>

[STEPS]
?. [DNase Treatment]
Take 10μL sample pre-DNase, mixed with  for analytical gel.
[2x formamide dye]
2x Formamide Dye can be made as follows: 20mL formamide2.2mL of 100mM (10mM final concentration) EDTAA spatula tip-ful of powdered xylenecyanolA spatula tip-ful of powderedbromophenol blue
?. [DNase Treatment]
Add to each 1 ml reaction.
[RNase-Free DNase]
?. [DNase Treatment]
Return samples to RNase-Free heat block and incubate at  for  .
37 °C
?. [DNase Treatment]
Spin down , ,  to pellet pyrophosphate.
4 °C
Centrifuge: 15000 34
?. [DNase Treatment]
Move 3mL to a new tube. Add .
[2x formamide dye]
?. [DNase Treatment]
Keep  until Gel Purification.
on ice
Reaction can be frozen at -80°C.
?. [RNA Elution]
Centrifuge , , ~.
4 °C
Centrifuge: 4000 33
Want 1mL each reaction.
?. [RNA Elution]
Split each into 3 Eppendorf tubes of .
333 µl
?. [RNA Elution]
Add 0.3 volumes  (100uL) to each sample.
[NaOAc]
?. [RNA Elution]
Resuspend each sample pellet in  - .
200 µl
[DEPC total]
?. [RNA Elution]
Place eppendorf with pellet on  heat block with the lid open and a kim-wipe on top for -.
37 °C
?. [RNA Elution]
Pipette off supernatant.
?. [RNA Elution]
Add   (3 volumes) to each sample.
[Chilled 100% EtOH]
?. [RNA Elution]
Centrifuge , , .
4 °C
Centrifuge: 20000 34
?. [RNA Elution]
Combine with other pellets of the same sample.
?. [RNA Elution]
Add  to wash pellet.
[70% EtOH]
?. [RNA Elution]
Pipette supernatant off.
?. [RNA Elution]
Centrifuge samples , , .
4 °C
Centrifuge: 20000 34
Do Fast Temp before to make sure it's at   as soon as samples go in.
?. [RNA Elution]
Chill in  at least .
-80 °C
?. [RNA Elution]
Nanodrop RNA.
?. [RNA Elution]
Label and put RNA at .
-80 °C
?. [Gel Purification]
Pre-run, 25 W for about   or until metal plate is warm.
?. [Gel Purification]
Heat denature samples at , .
65 °C
?. [Gel Purification]
Load each sample into separate wells.
?. [Gel Purification]
Take gel out and sandwich between saran wrap.
?. [Gel Purification]
Use UV shadowing to identify RNA bands.
?. [Gel Purification]
Highlight bands with sharpie.
?. [Gel Purification]
With clean razor blade, cut out bands and place in 50 ml Falcon tube.
?. [Gel Purification]
Crush gel pieces with RNase Free serological pipette.
?. [Gel Purification]
Add  , .
[DEPC]
[NaOAc]
Want: 5mL of DEPC/NaOAc per 1mL of IVT reaction and 1/30 NaOAc.
?. [Gel Purification]
Rock at , .
4 °C
?. [Gel Purification]
Cast large 15% Urea-PAGE gel:
[15% Urea-PAGE]
[TEMED]
[30% APS]
?. [Q5 PCR Amplification]
Prepare amplification reactions (x8) (plus 1.1x for pipette error) for each sgRNA.
[Oligo 1 Primer (10μM)]
[Oligo 2 Primer (10μM)]
[Oligo 3 Primer (10μM)]
[dNTPs (10mM)]
[Q5 Buffer]
[Q5 Polymerase]
[DEPC]
Amplification reactions of 8.8x are recommended, but the 1x Q5 PCR reaction is as follows (able to scale up or down): 1μL (10μM) Oligo 1 Primer1μL (10μM) Oligo 2 Primer 1μL (10μM) Oligo 3 Primer0.5μL (10mM) dNTPs5μL Q5 Buffer0.25μL Q5 Polymerase16.25μL DEPC
?. [Q5 PCR Amplification]
Run using PCR conditions as follows:1 cycle:,  (initial denaturation)35 cycles of:,  (denaturation),  (annealing),  (extension)1 cycle:,  (final extension)Hold at .
98 °C
98 °C
51 °C
72 °C
72 °C
4 °C
Reaction can be frozen at -20°C.
?. [Q5 PCR Amplification]
Run some reaction mix on an agarose gel to check PCR product size.
?. [In Vitro Transcription (IVT)]
From the 200μL PCR reactions (25μL x 8 tubes), split into 3 tubes of 66.6μL. Each tube will have a final volume of 1mL after IVT.
The PCR product doesn’t need to be cleaned before IVT.
?. [In Vitro Transcription (IVT)]
For each 1mL IVT reaction, add the following:  (from previous step)
[10x IVT buffer]
[5x NTPs]
[T7 Polymerase]
[RNase Inhibitor]
[DNA template (15ug)]
[DEPC]
?. [In Vitro Transcription (IVT)]
Incubate in RNase free heat block at , .
37 °C
?. [DNase Treatment]
Add  to each 1mL reaction.
[10x DNase Buffer]
?. [DNase Treatment]
Take 10μL sample post-DNase, mixed with  for analytical gel.
[2x formamide dye]
?. [Gel Purification]
Run at 45W for  to .
?. [RNA Elution]
Filter sample through 0.22μm Corning Filter into 50mL tubes and keep
on ice
?. [RNA Elution]
Move samples to 15mL Amicon 3kDa MWCO Concentrators.
?. [RNA Elution]
Repeat wash step: add  per pellet in sample.
[70% EtOH]
?. [RNA Elution]
Pipette off supernatant.
?. [RNA Elution]
Centrifuge , , .
4 °C
Centrifuge: 20000 34
?. [Gel Purification]
Centrifuge , , .
4 °C
Centrifuge: 20000 34