Intro.
What is the central dogma process?
Replication, transcription, translation
DNA --> RNA --> Protein
What type of vector do we use in Waksman?
What does PCR stand for?
Polymerase Chain Reaction
What does RD stand for?
Restriction Digest
A 3-base sequence of mRNA.
What is a codon?
What would be the complementary DNA strand of...
AGGGCTTATTCGCGTA
The complementary DNA strand would be...
TCCCGAATAAGCGCAT
Main difference between cDNA library and genomic library?
cDNA library: contains the DNA we want
genomic library: contains all types of DNA fragments - DNA we want and do not want
What monomers does taq polymerase use to synthesize new DNA?
dNTPs
May cut in multiple places on the plasmid (places we don't want it to cut)
What is the minimum amount of base pairs your sequence must be?
300 bp
What would be the complementary RNA strand of...
GGTTAACGCGTAGATC
The complementary RNA strand would be...
CCAAUUGCGCAUCUAG
What are the two restriction sites we use to place our insert?
1. SFiIA
2. SFiIB
(pay attention to spelling - lowercase i then uppercase I)
How many times is amplification repeated?
30x
What restriction site do we use?
PVUII
A 3-base sequence found on tRNA.
What is an anticodon?
In what kind of areas does DNA replication occur? Why?
Occurs in A-T rich areas. A-T bonds are easier to separate because they are only connected with two hydrogen bonds rather than three like C-G bonds.
When purifiying the mRNA, what is the name of the column the genetic material is passed through?
Oligo-dT column
Create the clone name for this imaginary person
- 8th clone
- Name: Kevin Thomas
- School number: 16
- Year: 2056-2057
16KT8.56
How many base pairs do you subtract from your RD gels? Why?
700: 350 on each side
The PVUII sites are further outside the insert than the SFiIA and SFiIB sites
DNA is an abbreviation for this term.
What is deoxyribonucleic acid?
Explain DNA Replication
initiation:
- begins at "oriC”
- helicase unwinds by breaking the hydrogen bonds between complementary base pairs
- topoisomerase proteins surround the unzipping strands and relax the twisting
- single strand binding proteins binds to the single DNA strands and prevents it from rewinding
- primers bind to the starting point of replication
elongation:
- DNA polymerase elongates DNA by attaching complementary nucleotides
- the leading strand (3’ to 5’) is replicated continuously while the lagging strand (5’ to 3’) is replicated discontinuously, forming okazaki fragments
termination:
- exonucleases remove RNA primers and replace with appropriate DNA bases
- nucleases “proofread” the DNA and fix any mistakes
- DNA ligase seals the okazaki fragments to create one whole strand
What are the two selectable markers? How do they work?
1. Ampr (Ampicillin Resistance)
Agar plate has ampicillin, plasmid has gene that is resistant to ampicillin, only bacteria with the gene will grow.
2. LacZ Gene
Inserting the insert breaks the LacZ gene. This gene would originally produce a blue color. By breaking it, the colony will instead be white instead of blue.
List all 5 principles of DNA
Soluble in water
Can be denatured and renatured
Negatively charged
Can be stained by ethidium bromide
Absorbs UV light
What are the three uses/components of loading dye?
1. EDTA chelates divalent cations that inactivates the restriction enzyme
2. Dyes “mark” the DNA, so you can track the progression through the gel
3. Glycerol makes the DNA denser than the buffer
The type of bond that holds bases together in the double helix.
What is a hydrogen bond?