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Plv312

Manufactured by Addgene
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The PLV312.3 is a lab equipment product. It is designed for use in laboratory settings. The core function of the PLV312.3 is to perform specific tasks required in a laboratory environment.

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Lab products found in correlation

Nebuilder hifi dna assembly master mix, by New England Biolabs (3 mentions) Pcdna3.1 zeo, by Thermo Fisher Scientific (2 mentions) Pcmv pe2 tagrfp bleor, by Addgene (2 mentions) Lenti grna puro, by Addgene (2 mentions) Ecori hf, by New England Biolabs (2 mentions) Lentiguide puro, by Addgene (1 mentions) Pcmv abemax p2a gfp, by Addgene (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)

3 protocols using plv312

1

Construction of pCMV-PE2-tagRFP-BleoR Plasmid

Cited 1 time
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pCMV-PE2 (Addgene #132775, a gift from David Liu1 (link)) was digested with EcoRI-HF (NEB). After digestion, the plasmid was dephosphorylated by rSAP (NEB), followed by gel extraction. tagRFP was PCR amplified from pLV312.3 (Addgene #119944)19 (link). tagRFP was Gibson assembled into pCMV-PE2 using 50 ng backbone and 2:1 ratio of tagRFP amplicon. Assembly was performed with NEBuilder HiFi DNA Assembly Master Mix (NEB). Assembled plasmids were transformed into NEB Stable Competent E. coli, resulting in pCMV-PE2-tagRFP. P2A-BleoR (Zeocin resistance) was PCR amplified from an in-house plasmid that originated from pcDNA™3.1/Zeo (Invitrogen). pCMV-PE2-tagRFP was PCR amplified to get linearized plasmid, with amplicon end downstream of tagRFP. Gibson assembly was performed as described previously, resulting in pCMV-PE2-tagRFP-BleoR (Addgene #192508). Lenti-p3-eGFP plasmid was produced by replacing EF1a-PuroR (MluI-HF and ApaI) on Lenti-gRNA-puro (Addgene #84752, a gift from Hyongbum Kim20 (link)) with p3-eGFP sequence.
Mathis N., Allam A., Kissling L., Marquart K.F., Schmidheini L., Solari C., Balázs Z., Krauthammer M, & Schwank G. (2023). Predicting prime editing efficiency and product purity by deep learning. Nature biotechnology.
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2

Construction of pCMV-PE2-tagRFP-BleoR Plasmid

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
pCMV-PE2 (Addgene #132775, a gift from David Liu1 (link)) was digested with EcoRI-HF (NEB). After digestion, the plasmid was dephosphorylated by rSAP (NEB), followed by gel extraction. tagRFP was PCR amplified from pLV312.3 (Addgene #119944)19 (link). tagRFP was Gibson assembled into pCMV-PE2 using 50 ng backbone and 2:1 ratio of tagRFP amplicon. Assembly was performed with NEBuilder HiFi DNA Assembly Master Mix (NEB). Assembled plasmids were transformed into NEB Stable Competent E. coli, resulting in pCMV-PE2-tagRFP. P2A-BleoR (Zeocin resistance) was PCR amplified from an in-house plasmid that originated from pcDNA™3.1/Zeo (Invitrogen). pCMV-PE2-tagRFP was PCR amplified to get linearized plasmid, with amplicon end downstream of tagRFP. Gibson assembly was performed as described previously, resulting in pCMV-PE2-tagRFP-BleoR (Addgene #192508). Lenti-p3-eGFP plasmid was produced by replacing EF1a-PuroR (MluI-HF and ApaI) on Lenti-gRNA-puro (Addgene #84752, a gift from Hyongbum Kim20 (link)) with p3-eGFP sequence.
Mathis N., Allam A., Kissling L., Marquart K.F., Schmidheini L., Solari C., Balázs Z., Krauthammer M, & Schwank G. (2023). Predicting prime editing efficiency and product purity by deep learning. Nature biotechnology.
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3

Generating AAV Constructs and mRNA

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The sequences of the AAV constructs used in this work were generated by using pLV302 and pLV312.3 (Addgene plasmid nos.119943 and 119944) where regions of interest were exchanged using NEBuilder HiFi DNA Assembly Master Mix (NEB no. E2621). Amino acid sequences are listed in Supplementary Note 1. PCR was performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs). pCMV_ABEmax_P2A_GFP was a gift from David Liu (Addgene plasmid no. 112101). lentiGuide-Puro was a gift from Feng Zhang (Addgene plasmid no. 52963). The coding sequence of ABEmax was cloned into the mRNA production plasmid behind a T7 promoter for mRNA production and into pET His6 LIC cloning vector (2Bc-T, Addgene plasmid no. 37236) for protein production.
Rothgangl T., Dennis M.K., Lin P.J., Oka R., Witzigmann D., Villiger L., Qi W., Hruzova M., Kissling L., Lenggenhager D., Borrelli C., Egli S., Frey N., Bakker N., Walker JA I.I., Kadina A.P., Victorov D.V., Pacesa M., Kreutzer S., Kontarakis Z., Moor A., Jinek M., Weissman D., Stoffel M., van Boxtel R., Holden K., Pardi N., Thöny B., Häberle J., Tam Y.K., Semple S.C, & Schwank G. (2021). In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nature Biotechnology, 39(8), 949-957.
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