in vitro gRNA transcription

Transcription with non-modified nucleotides


Responsive image

Linearize Vector


Use high-quality mini-prepped or maxi-prepped DNA for transcription. Starting with the pT7gRNA vector that has the cloned gRNA target site, linearized the plasmid with NotI (NEB #R3189S), preferably overnight. Note, it is important to ensure complete digestion, as even small amounts of undigested plasmid will result in the presence of transcripts seen as a smear on the denaturing gel migrating above the predicted size. The reaction can be optionally cleaned and then re-digested for 30 minutes the following morning. NotI generates a 5' overhang which prevents T7 polymerase re-association and transcription of the non-template strand.


Digest a couple 3-5μg of plasmid:

DNA	10μl
Buffer	5μl
H2O	31μl
NotI-HF	4μl

Purify reaction using Zymo Clean and Concentrator-5 (Zymo D4013), and elute with 10-12μl

Quantitate DNA using NanoDrop.


T7 reaction


Reagents:

HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050S)


Transcribe 1μg of linearized plasmid.

For example, if plasmid concentration is 200ng/μl:

Template DNA	5μl
NTP Buffer	10μl
H2O		13μl
T7 polymerase	2μl

Incubate at 37C for 4-16hrs


DNase I


Add 20µl to raise the final volume to 50μl with H2O

add 2μl DNase I

incubate at 37C for 15 minutes


Purification


Reagents:

Ambion MEGAclear Transcription Clean-Up Kit (AM1908)


Add 50µl to raise the final volume to 100μl with H2O

add 350μl of Binding buffer

add 250μl of 100% Et-OH

Mix and add to column


Pre-warm elution solution at 65C

Centrifuge at 10,000 X g for 30 sec

Wash 2X with 500μl Wash buffer

30 sec additional spin to remove trace Et-OH


Elute in two sequential elutions:

1st elution add 50μl Elution Buffer and incubate column at 65C for 10 minutes prior to centrifugation;

2nd elution add 50μl Elution Buffer and spin immediately.


Quantitate RNA using NanoDrop, and store RNA at -80°C.

(Expect concentration from 300-400 ng/μl to over 1μg/μl)


Denaturing gel


Run RNA on 10% (or greater) denaturing PAGE

This is to ensure that the RNA quality is good and/or there is no degradation, which appears as smears below the expected size, or undigested plasmid, which appears as large smears above the expected size. Run ~50ng of RNA along with RNA ladder and look for a band around ~110nt or higher. Note: several closely migrating distinct bands in this size range are possible due to potential RNA secondary structure.

Alternatively, run 1μl of 10-20ng on a Bioanalyzer RNA chip.

Transcription with modified nucleotides


Responsive image

Linearize Vector


Use high-quality mini-prepped or maxi-prepped DNA for transcription. Starting with the pT7gRNA vector that has the cloned gRNA target site, linearized the plasmid with NotI (NEB #R3189S), preferably overnight. Note, it is important to ensure complete digestion, as even small amounts of undigested plasmid will result in the presence of transcripts seen as a smear on the denaturing gel migrating above the predicted size. The reaction can be optionally cleaned and then re-digested for 30 minutes the following morning. NotI generates a 5' overhang which prevents T7 polymerase re-association and transcription of the non-template strand.


Digest a couple 3-5μg of plasmid:

DNA	10μl
Buffer	5μl
H2O	31μl
NotI-HF	4μl

Purify reaction using Zymo Clean and Concentrator-5 (Zymo D4013), and elute with 10-12μl

Quantitate DNA using NanoDrop.


T7 reaction


Reagents:

HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040S)

Pseudouridine-5'-Triphosphate

5-Methylcytidine-5'-Triphosphate


Transcribe 1μg of linearized plasmid.

For example, if plasmid concentration is 200ng/μl a standard nucleotide reaction:

Template DNA		5µl	
10X Reaction Buffer	1.5µl (0.75x final)
ATP (100mM)		1.5µl (7.5mM final)
GTP (100mM)		1.5µl (7.5mM final)
CTP (100mM)		1.5µl (7.5mM final)
UTP (100mM)		1.5µl (7.5mM final)
H2O			6µl
T7 polymerase		1.5µl

Incubate at 37C for 4-16hrs

To substitute modified nucleotides in a T7 reaction, it is best to first optimize the ratio of modified/un-modified nucleotide, as yields will typically be lower than standard nucleotides. 100% modified, 50% modified/50% unmodified, and 25% modified/75% unmodified are good strating points.


DNase I


To the T7 reaction, add the following:

70µl of H2O

10µl of 10X DNase Buffer

2μl DNase I

Incubate at 37C for 15 minutes


Purification


Reagents:

Ambion MEGAclear Transcription Clean-Up Kit (AM1908)


add 350μl of Binding buffer

add 250μl of 100% Et-OH

Mix and add to column


Pre-warm elution solution at 65C

Centrifuge at 10,000 X g for 30 sec

Wash 2X with 500μl Wash buffer

30 sec additional spin to remove trace Et-OH


Elute in two sequential elutions:

1st elution add 50μl Elution Buffer and incubate column at 65C for 10 minutes prior to centrifugation;

2nd elution add 50μl Elution Buffer and spin immediately.


Quantitate RNA using NanoDrop, and store RNA at -80°C.

(Expect concentration from 300-400 ng/μl to over 1μg/μl)


Denaturing gel


Run RNA on 10% (or greater) denaturing PAGE

This is to ensure that the RNA quality is good and/or there is no degradation, which appears as smears below the expected size, or undigested plasmid, which appears as large smears above the expected size. Run ~50ng of RNA along with RNA ladder and look for a band around ~110nt or higher (transcripts containing modified nucleotides will have altered mobility, and typically run slower due to increased molecular weight). Note: several closely migrating distinct bands in this size range are possible due to potential RNA secondary structure.

Alternatively, run 1μl of 10-20ng on a Bioanalyzer RNA chip.