ignore - Optional Tutorial 1: Molecular Tools Flashcards
In gel electrophoresis i) __________ DNA fragments travel faster so they appear closer to the ii) ___________ of the gel after samples are run.
A i) larger; ii) top
B i) smaller; ii) bottom
C i) larger; ii) bottom
D i) smaller; ii) top
B - The agarose polymer creates a mesh-like network in the agarose gel that impedes the movement of larger molecules so that smaller DNA fragments travel faster and further along the gel.
Imagine you have four samples of DNA labeled, A, B, C, and D. You perform a Southern Blot to determine which of these four samples contains a specific DNA sequence of interest. You run these samples side by side in separate lanes on the electrophoresis gel, transfer to a nitrocellulose membrane and incubate them with a probe for the sequence of interest. If only samples B and D contain the sequence of interest what will your final autoradiogram look like?
Because only samples B and D contain the sequence of interest, the probe will only bind the DNA in these lanes. DNA is present in all lanes but will only appear on the autoradiograph where the probe is bound. Therefore the autoradiogram will have a band in lanes B and D only.
Now imagine you run this experiment again but this time you are testing for two new sequences of interest, sequence 2 and sequence 3. You run these samples again on two electrophoresis gels, transfer and incubate each membrane with a separate probe; Probe 2 and Probe 3, complimentary to these two sequences of interest. If only samples A and C contain sequence 2, and only sample D contains sequence 3, what will your autoradiograms look like?
will only see bands in the A and C lane for probe 2 and lane D for probe 3 as these are the only probed sequences of interest
If the gene locus encoding the IgM heavy chain variable domain in mice has 90 V, 25 D, and 6 J segments, and the Lambda light chain variable domain has 75 V and 10 D segments, how many possible combinations exist for the variable region of the IgM/Lamda Ab?
= Heavy x light
= (V x D x J)(V x D)
= (90 x 25 x 6)(75 x 10)
= 10 125 000 combinations
If gene locus encoding the IgG heavy chain variable domain in rabbits has 60 V, 32 D, and 8 J segments, and the Kappa light chain variable domain has 86 V and 11 D segments, how many possible combinations for the variable region of the IgG/Kappa Ab are there?
= Heavy x light
= (V x D x J)(V x D)
= (60 x 32 x 8)(86 x 11)
= 14 530 560 combinations
Fill in the blanks: A mouse anti-GAPDH antibody is a ________ (primary/secondary) antibody that recognizes the antigen ________.
primary, GAPDH
Fill in the blanks: A goat anti-mouse antibody is a ________ (primary/secondary) antibody produced in ________.
secondary, goats
What is the name of the gel used in gel electrophoresis?
agarose gel
What is the purpose of gel electrophoresis?
to separate molecules via their size and charge
What does hybridization mean?
the combination of 2 complementary DNA strands that originate from different sources
what do dsDNA and ssDNA stand for?
double-stranded and single-stranded
What is the purpose of a southern Blot?
it is a blotting system to find a specific DNA sequence from a larger sample of DNA
What is the difference between the southern and northern blot?
southern detects a specific DNA sequence while northern detects a specific RNA sequence
what is an autoradiogram?
a diagram that only shows hybridized bands of DNA/RNA.
What is the purpose of Fluorescent in-situ hybridization?
it hybridizes whole chromosomes therefore it is used to produce karyotypes
What is the purpose of RNA in-situ hybridization?
this hybridizes mRNA transcript sequences allowing for the analysis of gene expression
What is a
a) restriction enzyme
b) restriction site
a) group of endonuclease that cleaves foreign DNA
b) site in which restriction enzymes cleave
most restriction sites are 4 to 8 Bp long and are considered palindromic. What does this mean?
when read 5’ to 3’ both strands have the same sequence
What is the difference between a blunt end and a sticky end? Which is more common?
blunt end is when the restriction enzymes cleave straight through the 2 complementary strands
sicky end is when the restriction enzyme cleaves both strands at different locations resulting in an overhang from both strands.
What are the 4 steps to DNA cloning?
- digestion = cleave plasmid at MCS and destroy DNA sample into fragments
- ligation = due to the sicky end of the plasmid being complementary to the denatured DNA and the presence of DNA Ligase the target fragment is inserted in the DNA
- transformation = mix the recombined plasmid w/ cells in order to encourage the cells to take in the recombinant plasmid
- selection = place the cells on a medium that contains a specific set of antibodies which will kill off any cells that do not contain the recombined plasmid
describe clone screening
the act of using gel electrophoresis to identify the recombinant plasmids from the non-recombinant plasmids
What basic form do all antibodies (Abs) come in?
a protein with 4 polypeptides in which 2 are heavy chains and 2 are light chains
what are the 3 domains of Ab’s? Which one accounts the most for the high specificity of Ab’s
1) constant
2) variable
3) hyper-variable - contains the most diversity
it is said that the antigen binding site binds to the epitope b/w an Ab and Ag (antigen) complex. Which site belongs to which molecule?
antigen binding site is on the Ab while the epitope is on the Ag
