Adenoviruses were prepared and amplified with the ViraPower Adenoviral Expression System (Thermo Fisher Scientific), as previously described [22 ]. The shRNAs of N4bp2l and LacZ were cloned into BLOCK-iT U6 entry vector (Thermo Fisher Scientific). The shRNA sequence for N4bp2l1 shRNA#1 was 5′-cacc GAATAACTATGAAGTTATA ttcaagaga TATAACTTCATAGTTATTC-3′ and for N4bp2l1 shRNA#2 was cacc GAAAGAATTGGATTGAAAT ttcaagaga ATTTCAATCCAATTCTTTC. The pENTR vector inserts were transferred into the adenovirus vector pAd/PL-DEST using the Gateway System (Thermo Fisher Scientific). The recombinant adenoviruses were purified using the Adenovirus Purification Miniprep Kit (Cell Biolabs) according to the manufacturer's protocol.
Adenovirus purification miniprep kit
Manufactured by Cell Biolabs
Sourced in United States
The Adenovirus Purification Miniprep Kit is a laboratory tool designed to isolate and purify adenoviral particles. The kit includes the necessary reagents and protocols to extract and concentrate adenovirus samples from cell culture or other sources.
Automatically generated - may contain errors
Lab products found in correlation
Gateway system, by Thermo Fisher Scientific (4 mentions)
Virapower adenoviral expression system, by Thermo Fisher Scientific (4 mentions)
Block it u6 entry vector, by Thermo Fisher Scientific (3 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo cloning kit, by Thermo Fisher Scientific (1 mentions)
Pad cmv v5 dest vector, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo vector, by Thermo Fisher Scientific (1 mentions)
4 protocols using adenovirus purification miniprep kit
1
Adenoviral Knockdown of N4bp2l1 and LacZ
2
Adenoviral Expression of Kbtbd11
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We subcloned the C-terminal 3xFLAG- and 6xHis-tagged full-length Kbtbd11 into the pENTR/D-TOPO vector (Thermo Fisher Scientific). The inserts of the pENTR-Kbtbd11-3xFLAG-6xHis vector were transferred into the pAd/CMV/V5-DEST vector using the Gateway system (Thermo Fisher Scientific). Adenoviruses were prepared and purified using the ViraPower Adenoviral Expression System (Thermo Fisher Scientific) and the Adenovirus Purification Miniprep Kit (Cell Biolabs), respectively, as described previously26 (link).
3
Adenovirus Production and Gene Silencing
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Adenoviruses were prepared using the ViraPower Adenoviral Expression System (Invitrogen), as previously described1,19, 20,21. PCR-amplified, C-terminal 6xHis and 3xFLAG tagged full-length Ildr2 was subcloned into the pENTR/D-TOPO vector using the pENTR Directional TOPO Cloning Kit (Invitrogen). Inserts of pENTR-Ildr2-6xHis-3xFLAG vector were transferred into the pAd/CMV/V5-DEST vector by the Gateway system (Invitrogen).
For the shRNAs of Ildr2 and LacZ were cloned into BLOCK-iT U6 entry vector (Invitrogen). The sequence of the shRNA for Ildr2 shRNA1 was: 5′- cacc GAAGAAGGTGGCCATGCTC acgtgtgctgtccgt GAGCATGGCCACCTTCTTC -3′ and Ildr2 shRNA2 was: cacc GCTGATTTCAAATCTTAGT gcgcttcctgtcacgc ACTAAGATTTGAAATCAGC. The inserts of pENTR shRNAs vectors were transferred into the adenovirus vector pAd/PL-DEST using the Gateway system (Invitrogen). The recombinant adenoviruses were purified by the Adenovirus Purification Miniprep Kit (Cell Biolabs).
For the shRNAs of Ildr2 and LacZ were cloned into BLOCK-iT U6 entry vector (Invitrogen). The sequence of the shRNA for Ildr2 shRNA1 was: 5′- cacc GAAGAAGGTGGCCATGCTC acgtgtgctgtccgt GAGCATGGCCACCTTCTTC -3′ and Ildr2 shRNA2 was: cacc GCTGATTTCAAATCTTAGT gcgcttcctgtcacgc ACTAAGATTTGAAATCAGC. The inserts of pENTR shRNAs vectors were transferred into the adenovirus vector pAd/PL-DEST using the Gateway system (Invitrogen). The recombinant adenoviruses were purified by the Adenovirus Purification Miniprep Kit (Cell Biolabs).
4
Adenoviral Expression System for Gene Manipulation
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Adenoviruses were prepared and amplified with the ViraPower Adenoviral Expression System (Invitrogen, Carlsbad, CA, USA), as previously described.11 Polymerase chain reaction (PCR)‐amplified, mouse Kbtbd11 complementary deoxyribonucleic acid was subcloned into the pENTR Directional TOPO vector (Invitrogen). The short hairpin ribonucleic acids (shRNAs) of Kbtbd11 and LacZ were cloned into BLOCK‐iT U6 entry vector (Invitrogen). The sequence of the shRNA for Kbtbd11 shRNA#1 was as follows: 5′‐cacc GGACATATGTGAAATCTGA ttcaagaga TCAGATTTCACATATGTCC‐3′, and Kbtbd11 shRNA#2 was as follows: cacc GCAAGTAAGTGACATTTAA ttcaagaga TTAAATGTCACTTACTTGC. Inserts of pENTR vectors were transferred into the adenovirus vectors pAd/CMV‐DEST or pAd/PL‐DEST using the Gateway system (Invitrogen). Recombinant adenoviruses were purified by the Adenovirus Purification Miniprep Kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's protocol.
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