1. Extract RNA using a commercial kit 2. DNase treatment using RNase free DNase 1U/ul from Promega to digest residual plasmid DNA 3. Use ABI TaqMan One-Step RT-PCR master mix […]
qRT-PCR for Lentivirus Titration
1. Make two primer probe sets U5-Psi: U5-Psi forward: TGTGTGCCCGTCTGTTGTGT U5-Psi reverse: GAGTCCTGCGTCGAGAGAGC U5-Psi probe: FAM-CAGTGGCGCCCGAACAGGGA-TAMARA WPRE: WPRE forward: CCGTTGTCAGGCAACGTG WPRE reverse: AGCTGACAGGTGGTGGCAAT WPRE probe: FAMTGCTGACGCAACCCCCACTGGT-TAMARA 2. Purify RNA from […]
Concentrate Lentivirus by Ultra-centrifugation
Ultracentrifuge: Beckman LE 80K Rotor: SW28 Tubes: #326823 autoclaved (polyallomer) 1. Pool all viral media into and filter through a 0.45μm HV Durapore membrane (Millipore #SCHVU01RE). 2. Place tubes into […]
Lentivirus Purification by Ultra-centrifugation
Before Starting: Required equipments and consumbles: 1. SW-28 rotor with buckets and caps (Kept in 4C Refrigerator in Centrifuge Room) 2. SW-28 thin walled ultra clear centrifuge tubes (Beckman # […]
Formulation of Lipofectamine 2000 and Lipofectin
LIPOFECTIN is a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE). LIPOFECTAMINE is a 3:1 (w/w) mixture of the polycationic lipid, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and DOPE.
Comparison of HSV, Adenovirus, AAV, Retrovirus and Lentivirus for Gene Delivery
HSV Adenovirus AAV Retrovirus Lentivirus Genomic Integration Episomal,100% Episomal,100% Episomal >90%, Integrated Integrated Cloning Capacity 150 kb for amplicon; 40 kb for replication defective HSV 8 kb for replication defective […]
Lentivirus Titration via qRT-PCR
The transduction unit (TU or IFU) of the lentiviral particles was estimated using the formula which 1TU=100 copies of viral genomic RNA. The physical copy numbers of the viral genomic […]
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