rDNA final Flashcards
In blue white test, what does host E. coli have?
carries the lacZ deletion mutant (lacZΔM15} which contains the ω-peptide
In blue white test, what does vector plasimd have?
plasmids used carry the lacZα sequence which encodes the first 59 residues of β-galactosidase, the α-peptide
What is formed when a plasmid containing the lacZα sequence is transformed into a lacZΔM15 cells?
a functional β-galactosidase enzyme
What is within the plasmid lacZα sequence of blue white test?
internal multiple cloning site (MCS)
How is the gene and thus production of α-peptide disrupted?
cut by restriction enzymes so that the foreign DNA may be inserted within the lacZα gene
What is X gal and what does it detect?
a colourless analog of lactose, detects active β-galactosidase
What are the seven steps of cloning
1) provide vector (plasmid)
2) provide insert
3) cut vector (plasmid)
4) ligate vector and insert
5) transform cells by introducing rDNA in to host cells
6) screen - check for blue and white
7) amplify - pick white, grow in media isolate DNA
What ends are catalyzed by DNA ligase?
3’ OH to 5’ PO4 to form phosphodiester bond
What are four rules for ligation?
1) small 10-20 micro L volue so ends not too dilute
2) for 2-6 kg vector use about 50-100 ng DNA
3) emperical - optimal insert
4) higher molar ration 1:1, 1:3, 1:10 vector
Name four applications of PCR?
1) make DNA for cloning on sequencing
2) screen DNA for sequence - diagnosis disease, screen for bacteria and viruses, match criminals to crimes
3) forensics, ie paternity teststing
4) rt PCR
5) reverse transcriptase PCR
What are the five components of PCR?
1) template DNA - genomic or cDNA
2) primers - foward to antisense, reverse to sense
3) nucleotides dATP, dTTP, dCTP, dGTP
4) DNA polymerase - Taq DNA
5) buffer with Mg - Taq cofactor
Four steps of PCR with temp/time
1) Denature 94-95 1 minute
2) Anneal 45-60, primer onto template
3) Extend 72, to the 3’ end
4) Repeat
What does denaturing mimic?
the function of helicase (at 95 C)
What does the forward primer bind to?
Antisense strand
What does the lower primer bind to?
Sense strand
From where does the extension of DNA begin?
At the primers’ 3’ end
What is typical size range for PCR?
300-2000 bpd
What is ideal primer size?
20-30 bp
What is ideal primer melting temp?
60 (50/50)
What is annealing temp?
4(G+C) + 2(A+T), about 5 C lower than MT
Detection methods using qPCR?
1) dsDNA binding
2) hybridization probes
How does dsDNA binding work?
incorporation SYBR green dye into dsDNA binding during PCR
How does a hybridization probe work?
two different probes labeled with differen fluorescent molecules
What does CT stand for?
threshold cycle
