Plasmid plus maxiprep kit
The Plasmid Plus MaxiPrep kit is a laboratory equipment designed for the purification of high-quality plasmid DNA from bacterial cultures. It utilizes a specialized column-based method to efficiently isolate plasmid DNA from large-scale samples.
Lab products found in correlation
12 protocols using plasmid plus maxiprep kit
Linear saRNA Synthesis and Purification
Lentiviral CRISPR Library Construction
Transient Transfection of Glycation Mutants
VEEV Self-Amplifying RNA Synthesis and Formulation
Schematic illustration of VEEV self-amplifying RNA (A), polymeric and lipid nanoparticle formulations (B) and pABOL chemical structure (C).
Plasmid DNA Isolation and Purification
Plasmid-based saRNA Production and Purification
SARS-CoV-2 Stabilized Pre-Fusion RNA Production
Ligation and Transformation of sgRNA Library
Synthesis and Purification of RNA Transcripts
were synthesized from constructs encoding firefly
luciferase in either mRNA with tobacco mosaic virus (TMV) 5′
and 3′ UTRs22 (link) or in saRNA with the
nonstructural proteins of the Venezuelan equine encephalitis virus
(VEEV), as previously described.20 (link) Plasmid
DNA (pDNA) was transformed into Escherichia coli,
cultured in 100 mL of LB with 50 μg/mL ampicillin (Gibco, Thermo
Fisher Scientific), and purified using a Plasmid Plus Maxiprep Kit
(QIAGEN). pDNA concentrations and purity were measured on a NanoDrop
One (Thermo Scientific). pDNA was linearized using SapI for 2 h at
37 °C and heat inactivated at 65 °C for 20 min. Uncapped
RNA transcripts, used for iteration A, were synthesized using 1 μg
of linearized DNA template in a MEGAScript T7 reaction (Invitrogen,
ThermoFisher Scientific), and capped RNA transcripts, used for iteration
B and validation of the optimized formulations, were synthesized using
750 ng of linearized DNA in a mMessage mMachine T7 reaction (Invitrogen,
ThermoFisher Scientific), according to the manufacturer’s protocol.
Transcripts were purified by overnight precipitation with LiCl at
−20 °C, pelleted by centrifugation at 13 000 × g and 4 °C for 15 min, washed one time with 70% EtOH,
centrifuged at 13 000 × g and 4 °C
for 5 min, and resuspended in Tris-EDTA buffer (Sigma-Aldrich), then
stored at −70 °C.
VEEV-Based Self-Amplifying RNA Production
derived from the Venezuelan Equine Encephalitis Virus (VEEV) encoding
either firefly luciferase (fLuc) or enhanced green fluorescent protein
(eGFP) was prepared using in vitro transcription.
pDNA was transformed in Escherichia coli and cultured
in 50 mL of LB with 1 mg/mL carbenicillin (Sigma–Aldrich, U.K.)
and isolated using a Plasmid Plus Maxiprep kit (QIAGEN, U.K.). pDNA
concentration and purity was measured on a NanoDrop One (ThermoFisher,
U.K.) and then linearized using MluI for 2 h at 37 °C and heat
inactivated at 80 °C for 20 min. Uncapped in vitro RNA transcripts
were synthesized using 1 μg of linearized DNA template in a
MEGAScript reaction (Promega, U.K.), according to the manufacturer’s
protocol. Transcripts were then purified by overnight LiCl precipitation
at −20 °C, pelleted by centrifugation at 14 000
rpm for 20 min, washed once with 70% EtOH, centrifuged at 14 000
rpm for 5 min, and then resuspended in UltraPure H2O. Purified
transcripts were then capped using the ScriptCap m7G Capping System (CellScript, Madison, WI, USA) and ScriptCap 2′-O-Methyltransferase
Kit (CellScript, Madison, WI, USA) simultaneously, according to the
manufacturer’s protocol. Capped transcripts were then purified
again by LiCl precipitation, resuspended in ultraPure H2O, and stored at −80 °C until use.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!