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Biology Lab Techniques
Immunology 150L
30
Biology
7th Grade
04/27/2010

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Term
Southern Blot
Definition
Visualizes chagnes in the arrangement of genomic DNA in one cell type compared to another based on mobility on gel electrophoresis
Term
How to compare rearranged vs unrearranged DNA
Definition

Clone DNA fragment containing rearrange gene

2) restriction map - to determine how it differes from unrearranged DNA

3)subclone - a smaller fragment containing site of recombination

4) Sequence - the rearranged DNA compare to sequence of unrearranged DNA, using bioinformatics likBLAST

Term
PCR
Definition

Polymerase Chain Reaction

Amplifies rearranged DNA from populations of cells

2) gel electrophoresis can be used to determien the types of rearrangement that occcur in a different cell type

Term
Why purify genomic DNA?
Definition

Can tell you the stages of immunological development

 

Term
Triton-X-100
Definition
nonionic detergent dissolves the plasma membrane of the cells while leaving the nuclear membrane intact
Term
tween-20
Definition
denatures proteins (such as DNases) and lyses nuclear membranes
Term
proteinase K
Definition

digests proteins

 

Term
How to precipitate DNA?
Definition
use isopropanol, it's insoluble and precipitates
Term
TE
Definition

Tris-EDTA a weak buffer with EDTA that traps metals and thus inhibits enzymes like DNases

Used commonly when storing protein/dna to save them from being degraded by trace enzymes

Term
How is DNA destroyed?
Definition

pH extremes

enzymes

shear forces

Term
There are 7 antigen receptor loci in lymphocytes, name them
Definition

T cells

alpha beta gamma sigma

B cells

kappa lambda

Term
RAG recombinase
Definition
catalyzes V-D-J rearrangement
Term
RSS
Definition

recombination signal sequence

heptamer

spacer (12/23)

nonamer

Rearrangement occurs adajacent to heptamer sequence

 

V's flanked 3' with RSS

D's Flanked both sides

J's flanked 5' with RSS

 

 

Term
RSS recombination
Definition
only RSS with opposite spacer sizes can rearrange together
Term
Heavy Chain Diversity
Definition

100V

13D

4J

Term
How does VDJ recombination imprecise?
Definition

Exact site of recombination varies

TdT (terminal transferase)- can add a few random base pairs at the site of joining

 

Term
Light Chain joins which two segments?
Definition

V and J

Also, TdT does not add N regions to light chains rearranging segments

Term
What causes the loss of nucleotides between rearranging segment DNA?
Definition
Exonucleases cause for more diversity between V-D and D-J
Term
What is a good 260/280 ratio?
Definition
>1.6
Term
How to calculate concentration of a sample of DNA?
Definition

If ratio is >1.6

Then concentration is OD x 50ug/ml = concentration of DNA

Term
One unit of restriction enzyme is?
Definition
one unit of restriction enzyme is defined by the amount nccessary to cut 1 ug of plasmid DNA in one hour
Term
What enables you to see DNA on a gel using UV light?
Definition
EtBr
Term
How do you Denature DNA?
Definition
NaOH
Term
DNA Complexity
Definition
total number of nonrepeating base-pairs inteh sample
Term
Southern blot sensitivity
Definition
1x10^6 copies for one band
Term
AP
Definition
catalyzes NBT and X-phosphate
Term
IPTG
Definition
inducer of lacZ gene
Term
x-Gal
Definition
substrate that turns blue
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