Methods Flashcards
How do TOPO vectors work?
Vaccinia virus DNA topoisomerase I is covalently bound to the 3´ end of each DNA strand following CCCTT. The isomerase ligates the free ends of the blunt DNA to each end of the TOPO vector. Positive clones are ensured by disruption of the lethal gene ccdB.
CAG promoter
Used in:
All envelope plasmids.
(C) the CMV early enhancer element,
(A) the promoter, the first exon and the first intron of chicken beta-actin gene,
(G) the splice acceptor of the rabbit beta-globin gene
CMV promoter
Used in:
Genome and rev plasmids.
CBA promoter
Used in:
GagPol plasmid.
Comprises:
Advantages:
How does the nanodrop work?
Calibrate using nucleotide free water.
260/280 gives a measure of purity.
Transmittance at set wavelengths gives a measure of nucleotide concentration compared to an internal assay.
Lux2
Encodes luciferase - enzyme requires luciferin.
Rev protein
Used for activating the translation of transgenes e.g. EGFP.
Purpose of PEIpro? Why add it to the DNA?
Cationic polymer that binds DNA. The neutral complex can more easily pass through cell membranes.
Why use FreeStyle?
Specifically developed for serum-free, suspension culture.
Fetal calf serum
Bits of cow
Sodium azide
Used for:
Antibody staining to prevent antigen internalisation.
Bovine serum albumin
Used to block stick proteins on the plasma membrane.
D-PBS
Salty stuff
The need for a three-stage staining procedure?
Flexibility – may be used with a variety of detection reagents, choose the second step reagent to suit your experiment.
Signal Amplification – useful for low-abundance antigens. Allows detection of poorly expressed antigen.
Why use PFA?
Fixative - slows the formation of covalent bonds in aqueous solution. Fixation for too long may lead to antigen internalisation.
Reason for adding Na+ butyrate?
Added to virus producer cells @24hrs post transfection.
Causes histone deacetylation and therefore helps drive expression in virus producer cells.
Reason for the harvest times?
Optimised
Filter sets used
UV for DAPI
FITC for Alexa 488
FL1 for EGFP
FL4 for Alexa 467
Polybrene
Polybrene is a cationic polymer used to increase the efficiency of infection by neutralizing the charge repulsion between virions and sialic acid.
The need for a precise quantity of cells for the FVT?
Did
How to design DNA sequencing primers
Design both forward and reverse (3’-5’) primers flanking the insert.
Sequencing further from the primer becomes less reliable. *0-150 bases away is most accurate. High GC content is desirable. Avoid primer sites that repeat.
NanoSight
Can monitor single particles and even fluorescently tagged ones.
Works on the principles of Brownian motion and light scattering.
What are HEKT cells?
The cells can be grown serum-free. Also, the expression of the SV40 large t antigen allows replication of plasmids with the SV40-origin
