Aug 21, 2018
Version 4
Protocol for in vitro transcription of DNA oligos by T7 polymerase V.4
- Audrey Lapinaite1,
- Sam Sternberg1
- 1University of California, Berkeley
- The Center for Genome Editing and Recording
Protocol Citation: Audrey Lapinaite, Sam Sternberg 2018. Protocol for in vitro transcription of DNA oligos by T7 polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.ssqeedw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2018
Last Modified: August 21, 2018
Protocol Integer ID: 14896
Abstract
Attachments
Guidelines
Template design:
T7 polymerase requires a double-stranded promoter region for efficient template binding, but can then transcribe a single-stranded DNA template. Therefore, the following protocol uses an approach, whereby two oligos are annealed in order to generate a dsDNA promoter, followed by a single-stranded overhang that contains the reverse complement of the desired RNA sequence.
The reverse complement of the desired RNA sequence is ordered as a DNA oligo (assuming the length does not exceed ~80 nt), to which the reverse complement of the T7 promoter is appended at the 3’ end Example template:
A separate, synthetic DNA oligo is ordered that contains the T7 promoter in the forward direction, which can be annealed with the above construct: T7oligo: 5’-TAATACGACTCACTATA-3’
Note: according to Ambion, for transcription of synthetic oligonucleotides, only the -17 to -1 positions of the promoter need to be double-stranded. This oligo takes this into account.
Materials
STEP MATERIALS
5x transcription buffer
1.5 nmol T7oligo
1.0 nmol template
DEPC H2O
5N NaOH
1N NaOH
RNase-free DNasePromega
EtOH
Silanization Solution I
15% urea polyacrylamide gel
40% Acrylamide-bisacrylamide (19:1)
Urea
Water
10% APS
1x TBE
1x TBE buffer
RNase-free water
Corning Spin-X filter tube
10x NEBuffer 3
CIP (10 U/μl)New England Biolabs
Phenol-chloroform solution
5x transcription buffer
1.5 nmol T7oligo
1.0 nmol template
DEPC H2O
5N NaOH
1N NaOH
RNase-free DNasePromega
EtOH
Silanization Solution I
15% urea polyacrylamide gel
40% Acrylamide-bisacrylamide (19:1)
Urea
Water
10% APS
1x TBE
1x TBE buffer
RNase-free water
Corning Spin-X filter tube
10x NEBuffer 3
CIP (10 U/μl)New England Biolabs
Phenol-chloroform solution
Protocol materials
10% APS
Silanization Solution I
40% Acrylamide-bisacrylamide (19:1)
Corning Spin-X filter tube
1N NaOH
Corning Spin-X filter tube
5x transcription buffer
1.5 nmol T7oligo
DEPC H2O
1x TBE
RNase-free DNasePromega
10x NEBuffer 3
RNase-free water
1N NaOH
DEPC H2O
RNase-free water
1.5 nmol T7oligo
1.0 nmol template
EtOH
Phenol-chloroform solution
EtOH
Urea
Urea
1x TBE buffer
CIP (10 U/μl)New England Biolabs
Silanization Solution I
5x transcription buffer
1x TBE
CIP (10 U/μl)New England Biolabs
1.0 nmol template
Phenol-chloroform solution
10% APS
5N NaOH
15% urea polyacrylamide gel
Water
5N NaOH
RNase-free DNasePromega
15% urea polyacrylamide gel
40% Acrylamide-bisacrylamide (19:1)
Water
10x NEBuffer 3
1x TBE buffer
5x transcription buffer
1.5 nmol T7oligo
1.0 nmol template
DEPC H2O
5N NaOH
1N NaOH
RNase-free DNasePromega
EtOH
Silanization Solution I
15% urea polyacrylamide gel
40% Acrylamide-bisacrylamide (19:1)
Urea
Water
10% APS
1x TBE
1x TBE buffer
RNase-free water
Corning Spin-X filter tube
CIP (10 U/μl)New England Biolabs
10x NEBuffer 3
Phenol-chloroform solution
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warnings and hazard information.
Before start
Stock solutions:
| 5x transcription buffer: | 10 ml | |
| 150 mM Tris-Cl, pH 8.1 | 1.5 ml | |
| 125 mM MgCl2 | 1.25 ml | |
| 0.05% Triton X-100 | 50 µl | |
| 10 mM spermidine | 100 µl | |
| H2O | to 10mL | |
| store at -20 °C |
| 10x TBE | 1 L | |
| Tris base | 108 g | |
| Boric acid | 55 g | |
| 0.5 M Na2EDTA pH 8.0 | 40 ml | |
| H2O | to 1 L |
| 0.5 M Na2EDTA | 0.5 L | |
| Disodium ethylenediaminetetraacetate 2H2O | 93 g | |
| NaOH to get pH 8.0 | 10 g | |
| H2O | to 500 ml | |
| autoclave |
| 10 % (w/v) APS | 50 ml | |
| Ammonium persulfate | 5 g | |
| H2O | to 50 ml | |
| store at -20 °C |
| 1 M spermidine | ||
| Spermidine | 0.7 g | |
| H2O | to 5 ml | |
| store at -20 °C |
| 5N NaOH | 25 ml | |
| NaOH | 5 g | |
| H2O | to 25 ml |
| 1N NaOH | 20 ml | |
| 5N NaOH | 5 ml | |
| H2O | to 20 ml |
| 10 % Triton X-100 | 10 ml | |
| Triton X-100 | 1 ml | |
| H2O | 9 ml | |
| store at -20 °C |
| 1% (w/v) bromophenol blue | 10 ml | |
| bromophenol blue | 100 mg | |
| H2O | to 10 ml |
| 1% (w/v) xylene cyanol | 10 ml | |
| xylene cyanol | 100 mg | |
| H2O | to 10 ml |
| 2x RNA Loading Dye | 30 ml | |
| 95% formamide | 28.5 ml | |
| 1 mM EDTA | 60 μl of 0.5 M | |
| 0.025% SDS | 75 μl of 10% | |
| 0.025% bromophenol blue | 750 μl of 1% | |
| 0.01% xylene cyanol | 300 μl of 1% | |
| store at -20 °C |
Preparing 10 uM hybridized template
Preparing 10 uM hybridized template
Mix the following in a final volume of 100 µl:
20 µl 5x transcription buffer
1.5 nmol T7oligo (15 uM final)
1.0 nmol template (10 uM final)
DEPC H2O to 100 µl
5x transcription buffer
1.5 nmol T7oligo
1.0 nmol template
DEPC H2O
Preparing 10 uM hybridized template
Preparing 10 uM hybridized template
Heat at 70-80 °C for 2 minutes.
70 °C Heating
00:02:00 Heating
Slow cool on bench-top.
Use immediately or store at -20 °C.
Note
For some applications, prior gel purification of the DNA template may be desirable. However, for my transcriptions, the oligos are not very long and I immediately gel purify the RNAs after transcription, so purity of the DNA oligo is not a major concern.
-20 °C Storage
Preparation of NTPs
Preparation of NTPs
Add 500 μl of RNase-free H2O to the 70-80 mg of each NTP.
500 µL RNase-free H2O
Bring pH of each NTP to 6.8-7.2 by adding 5 µl of 5N NaOH followed by 10-90 µl of 1N NaOH. Monitor pH in the stripes with 6.5-10 range. Most adjustment will be needed for ATP, least - for UTP.
5N NaOH
1N NaOH
Measure absorbance of 500 fold dilution: 3 µl to 1.5 ml of H2O.
ATP λmax 259 nm, εmax 1.59 × 104 cm-1 M-1
CTP λmax 271 nm, εmax 0.9 × 104 cm-1 M-1
GTP λmax 253 nm, εmax 1.37 × 104 cm-1 M-1
UTP λmax 259 nm, εmax 1.59 × 104 cm-1 M-1
Calculate concentrations and required dilutions to reach 100 mM concentration each.
Transcription reaction
Transcription reaction
Mix the following in a 100 μl reaction:
| Final concentrations: | ||
| 20 μl 5X transcription buffer | (1X) | |
| 5 μl 100 mM ATP | (5 mM) | |
| 5 μl 100 mM CTP | (5 mM) | |
| 5 μl 100 mM GTP | (5 mM) | |
| 5 μl 100 mM UTP | (5 mM) | |
| 1 μl 1M DTT | (10 mM) | |
| 0.1 μl 1 mg/mL pyrophosphatase (Roche) | (1 ug/ml) | |
| 10 μl 1 mg/mL T7 polymerase | (100 ug/ml) | |
| 10 μl 10 uM hybridized template | (1 uM) | |
| DEPC H2O to 100 μl |
Note
My typical final yields on transcription reactions of this size are roughly 30 μl of 10-20 uM (300-600 pmol). For my biochemical purposes, this is more than enough material. However, the above reaction can easily be scaled up.
5 µL 100 mM ATP
5 µL 100 mM CTP
5 µL 100 mM GTP
5 µL 100 mM UTP
1 µL 1M DTT
0.1 µL 1 mg/mL pyrophosphatase (Roche)
10 µL 1 mg/mL T7 polymerase
Incubate at 37 °C, overnight.
37 °C Incubation
16:00:00 overnight incubation
DNase treat:
- Add 5 units (5 μl) RNase-free DNase directly to reaction.
- Incubate at 37 °C, 30 minutes.
RNase-free DNasePromega
5 µL RNase-free DNase
37 °C Incubation
00:30:00 Incubation
Note
This step is optional and may be omitted.
Add equal volume (100 μl) gel loading buffer directly to transcription reaction.
100 µL Gel loading buffer
Note
My RNAs run very close to the xylene cyanol dye. Therefore, I use a gel loading buffer that contains only bromophenol blue.
Gel-purify transcribed RNAs on a medium thickness, denaturing urea-polyacrylamide gel.
Note
For my RNAs (~30 nt) I use a 15% gel. At this polyacrylamide concentration, with RNA from a 100 μl transcription reaction, I get excellent separation of the desired RNA length from n+1 and n-1 contaminants (these arise from template-independent addition of nts by T7 polymerase; and synthetic template oligos that are not the full length). Optimization of the gel running time and acrylamide concentration will be necessary for gel-purifying RNAs of different length.
Gel-purification
Gel-purification
PAGE Fragment Resolution: Denaturing Conditions (6M Urea)
Clean glass plates with EtOH and coat with 200 μl Silanization Solution I.
EtOH
Silanization Solution I
200 µL Silanization Solution I
Assemble glass plates.
Prepare 15% urea polyacrylamide gel.
15% urea polyacrylamide gel
Denaturing Urea Polyacrylamide Gel (300 ml for a gig gel: 35 x 25 cm)
| 10% | 12.5% | 15% | ||
| 10x TBE | 30 ml | 30 ml | 30 ml | |
| 40% Acrylamide-bisacrylamide (19:1) | 75 ml | 93.75 ml | 112.5 ml | |
| Urea | 108 g | 108 g | 108 g | |
| Water | 192 ml | 173 ml | 155 ml | |
| Before casting add: | ||||
| 10% APS | 1.5 ml | 1.5 ml | 1.5 ml | |
| TEMED | 600 µl | 600 µl | 600 µl |
After addition of APS and TEMED to Urea-Polyacrylamide mixture pour the gel immediately.
40% Acrylamide-bisacrylamide (19:1)
Urea
Water
10% APS
Place comb and polymerize for 60 min.
01:00:00 Polymerize
Assemble electrophoresis apparatus.
Remove comb.
Fill inner and outer buffer chambers with 1x TBE.
1x TBE
Rinse wells with 1x TBE buffer.
1x TBE buffer
Pre-run the gel for 30 min to heat-up and remove remaining urea. Set constant P=40 W,
3000 V, 400 mA.
00:30:00 Pre-running gel
Load sample.
Run P=40 W, 3000 V, 400 mA for around 3 h, until the bromophenol blue band is migrating in the last 1⁄4 of the gel.
03:00:00 Running gel
Remove one of the glass plates and cover the gel with saran wrap. Flip. Remove another glass plate and cover all gel with saran wrap.
UV shadow band and excise with sterile razor and place in Falcon tube.
Break gel slices with p-1000 tip.
Crush and soak gel slices in equal volume of RNase-free water.
RNase-free water
Elute RNA overnight by rocking at 4 °C.
4 °C Eluting RNA
Next day
Next day
Filter out gel pieces by spinning sample through Corning Spin-X filter tube. Centrifuge at top speed for 5 minutes, 4 °C.
4 °C Centrifugation
00:05:00 Centrifugation
Note
I cut the top off a P-1000 tip so that I can pipette as much as possible of the water-gel slurry into the Corning filter tube.
Corning Spin-X filter tube
Add 30 μl 10x NEBuffer 3 to each sample, then 1 μl CIP (10 U/μl). Incubate at 37 °C for one hour.
10x NEBuffer 3
30 µL 10x NEBuffer 3
CIP (10 U/μl)New England Biolabs
1 µL CIP
37 °C Incubation
01:00:00 Incubation
Note
This step is only required if removal of the 5’ triphosphate is required (e.g. if the RNA will subsequently be 5’-radiolabeled with T4 PNK.)
Phenol/chloroform extract with 300-500 μl phenol-chloroform solution, pH 8.0.
Phenol-chloroform solution
300 µL Phenol-chloroform solution
Ethanol precipitate the RNA, by adding 1/10 volume 3 M NaOAc (pH 5.2), 1/100 volume 100x linearized acrylamide and 3 volumes 100% ethanol. [described in step 35-40 below]
Incubate at -80 °C, at least 1 hour
-80 °C Incubation
01:00:00 Incubation
Spin down at top speed, 20 minutes, 4 °C.
4 °C Spinning down
00:20:00 Spinning down
Remove supernatant.
Wash pellet with 70% ethanol.
Spin down again and remove supernatant.
Dry pellets in speed vacuum, ~10 minutes.
00:10:00 Drying pellets
Resuspend pellets in 30 μl DEPC H2O (or buffer) and store at –20 °C.
300 µL DEPC H2O (or buffer)
-20 °C Storage