T7 promoter sequence
T7 promoter oligo, we added this ologo sequence to the 5' end of oligo DNA template sequence for shRNA transcription. So that shRNAs can be generated in large scale through in vitro transcription system.
We added T7 promoter to the 5-prime end of each shRNA template sequence for in vitro transcription of the shRNAs,
T7 promoter is originally obtained from bacteriophage T7, it is specific for bacteriophage T7 RNA polymerase. Bacteriophage T7 (or the T7 phage) is a bacteriophage, a virus that infects susceptible bacterial cells, that is composed of DNA and infects most strains of E. coli. The T7 promoter is widely used for in vitro transcription of a DNA template. The T7 promoter is also commonly used in E. coli expression studies.
1) DNA Oligo Template Design
For primer 1, convert the sense strand of the siRNA sequence to the corresponding DNA sequence, add a 17 base T7 promoter sequence (TAATACGACTCACTATA) to the 5’end of the DNA sequence, add an 8 base loop sequence to the 3’-end of the DNA sequence. For primer 2, add the antisense sequence complementary to the loop sequence to the 3’-end of the DNA sequence. add 2 AA’s to the 5’-end of the Primer 2 oligo. 5 pairs of DNA Oligo (Table 1) were ordered.
|Primers for DNA oligo template||Primers|
|ARK-shRNA-1||P1: 5’TAATACGACTCACTATAGGAGTTCGATGCCGTTAAGGACTTGCTTC 3’ |
P2: 5’AAGGAGTTCGATGCCGTTAAGGAGAAGCAAG 3’
|GLS-shRNA-1||P1: 5’TAATACGACTCACTATAGGTCGGAGAAACCGATTGGGACTTGCTTC 3’ |
P2: 5’AAGGTCGGAGAAACCGATTGGGAGAAGCAAG 3’
|GLS-shRNA-2||P1: 5’TAATACGACTCACTATAGGCGTACATCGATCAACGTCCTTGCTTC 3’ |
P2: 5’AAGGCGTACATCGATCAACGTCGAAGCAAG 3’
|ALR-shRNA-1||P1: 5’TAATACGACTCACTATAGGTCTTGTCAAATCAGTAGGACTTGCTTC 3’ |
P2: 5’AAGGTCTTGTCAAATCAGTAGGAGAAGCAAG 3’
|ALR-shRNA-2||P1: 5’TAATACGACTCACTATAGGACCGTCCGTTCGCCAAGCCCTTGCTTC 3’ |
P2: 5’AAGGACCGTCCGTTCGCCAAGCCGAAGCAAG 3’
2) Fill-in reaction to generate ARK-shRNA-1 transcription templates
Each fill-in Reaction was set up with two Oligos
1.0 µl P1 Oligo (100 pmoles)
1.0 µl P2 Oligo (100 pmoles)
2.0 µl 10 x buffer 2 (NEB)
0.5 µl 50 X dNTPs (10 mM)
0.5 µl Klenow Fragment exo– DNA Polymerase (5 U/ ml)
15 µl RNase-Free Water
20 µl Total reaction volume, incubate the reaction mixtures for 2 hours at 37ºC, then 25 min at 75 ºC, cool at room temperature for 2 minutes.
The integrity of ALR-shRNA-1 templates was identified through 3% agarose gel eletrophoresis (Fig. 1). Agarose gel electrophoresis showed that the ALR-shRNA-1 template was successfully generated. Sharp bands can be seen on the gel, and the size of the bands is around 69 bases, which is the correct size.
3) In vitro transcription
1. in vitro transcription reaction was set up using the prepared template.
10.7 µl RNase-Free Water
2.0 µl Fill-In Reaction product
2.0 µl 10 x T7 RNA Polymerase Buffer (NEB)
2.8 µl 100mM MgSO4 (NEB)
1.0 µl NTP Mix (80 mM each NTP)
1.5 µl T7 RNA Polymerase (50 U/ µl)
20 ul Total reaction volume
2. Incubate the reaction mixtures for 2-3 hours at 37ºC.
3. Add 1 µl RNase-Free DNase I (1 Unit/ml) to remove the DNA template, 37ºC 15 min.
4. Heat the reaction mixtures for 15 minutes at 70ºC to inactivate the enzyme.
5. Extract with Phenol/Chloroform.
a. Add 100 µl RNase-Free Water to dilute the reaction.
b. Add 120 µl phenol/chloroform and vortex briefly to mix.
c. Spin in a microfuge for 1 minute at full speed.
d. Carefully pipette off the top aqueous phase and transfer to a clean tube.
6. Precipitate the shRNA.
a. To the recovered aqueous phase, add 1/10 vol. of 3 M Sodium Acetate (pH 5.2).
b. Add 2.5 volumes of 95-100% ethanol.
c. Incubate for 15 minutes on ice.
d. Pellet the shRNA in a microfuge by spinning at full speed for 15 minutes.
e. Remove the supernatant.
f. Carefully wash the pellet once with 70% ethanol.
g. Air dry the pellet for only 2-5 minutes.
7. Add 100 µl of the 1 X Annealing Buffer to the shRNA pellet and resuspend the shRNA.
The procedure of the shRNA in vitro transcription system is illustrated in Fig. 4-2.
The integrity of shRNAs were identified through 3% agarose gel eletrophoresis (Fig. 3) Agarose gel electrophoresis showed that the 5 shRNAs were successfully transcribed. Sharp bands of around 52 bases in length were detected on the gel, the size of the bands is correct.
Dunn, J. J.; Studier, F. W. (1983). "Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements". Journal of Molecular Biology. 166 (4): 477
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