Methods

Question 1

How do TOPO vectors work?

Answer 1

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.

Question 2

CAG promoter

Answer 2

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

Question 3

CMV promoter

Answer 3

Used in:

Genome and rev plasmids.

Question 4

CBA promoter

Answer 4

Used in:
GagPol plasmid.

Comprises:
Advantages:

Question 5

How does the nanodrop work?

Answer 5

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.

Question 6

Lux2

Answer 6

Encodes luciferase - enzyme requires luciferin.

Question 7

Rev protein

Answer 7

Used for activating the translation of transgenes e.g. EGFP.

Question 8

Purpose of PEIpro? Why add it to the DNA?

Answer 8

Cationic polymer that binds DNA. The neutral complex can more easily pass through cell membranes.

Question 9

Why use FreeStyle?

Answer 9

Specifically developed for serum-free, suspension culture.

Question 10

Fetal calf serum

Answer 10

Bits of cow

Question 11

Sodium azide

Answer 11

Used for:

Antibody staining to prevent antigen internalisation.

Question 12

Bovine serum albumin

Answer 12

Used to block stick proteins on the plasma membrane.

Question 13

D-PBS

Answer 13

Salty stuff

Question 14

The need for a three-stage staining procedure?

Answer 14

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.

Question 15

Why use PFA?

Answer 15

Fixative - slows the formation of covalent bonds in aqueous solution. Fixation for too long may lead to antigen internalisation.

Question 16

Reason for adding Na+ butyrate?

Answer 16

Added to virus producer cells @24hrs post transfection.

Causes histone deacetylation and therefore helps drive expression in virus producer cells.

Question 17

Reason for the harvest times?

Answer 17

Optimised

Question 18

Filter sets used

Answer 18

UV for DAPI
FITC for Alexa 488

FL1 for EGFP
FL4 for Alexa 467

Question 19

Polybrene

Answer 19

Polybrene is a cationic polymer used to increase the efficiency of infection by neutralizing the charge repulsion between virions and sialic acid.

Question 20

The need for a precise quantity of cells for the FVT?

Answer 20

Did

Question 21

How to design DNA sequencing primers

Answer 21

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.

Question 22

NanoSight

Answer 22

Can monitor single particles and even fluorescently tagged ones.
Works on the principles of Brownian motion and light scattering.

Question 23

What are HEKT cells?

Answer 23

The cells can be grown serum-free. Also, the expression of the SV40 large t antigen allows replication of plasmids with the SV40-origin