BCH5413 Exam 1 Questions and Answers
Correct
Incorrect
1 of 100
Term
Explain the steps of site directed mutagenesis
Give this one a try later!
plate with bacterial lawn
lay nylon membrane over plate to lift off replica of plaques
membrane acts like Southern blot membrane
Blocking with nonspecific DNA, and using a transfer with a lumescent probe that will
bind and light up sequences of interest
detect with autoradiography
,minimum base pairs required for single primer self-dimerization = 5bp
minimum bp for hairpin: 4bp
consider:
hybridization to other sequences = check via BLAST
secondary structure and Primer interactions
Is it G:C or A:T at 3' end - depends on your application
3' deltaG
Melting temperature differences
4 main types:
1. Ligation-mediated mutagenesis
2. PCR/Primer Extension mutagenesis
3. QuikChange mutagenesis
4. Inverse PCR mutagenesis
STEPS
1. Need plasmid: most replicated by E coli
2. Need ORI, MCS, Ap^r
3. plasmid transformed into E. coli
4. isolate small amount of plasmid DNA
5. screen plasmid DNA for mutation by 1: restriction site or 2: southern
hybridization
6. screen all mutated segments
NEED to DESTROY RESTRICTION SITE
EcoRI cuts at a site, digests, Klenow + dNTPs, T4 DNA ligase to put back
together
, Negative: for PCR, No template. For Ligation, no ligase. For DpnI, undigested
plasmid
Positive: For PCR, Control Plasmid with Primers. For Ligation, plasmid cut with 1
enzyme and ligase. For DpnI, control plasmid amplified with control primers
Don't know?
2 of 100
Term
State 2 ways in which you can increase the transfer efficiency of DNA
to a blot.
Give this one a try later!
1. Soak the gel in dilute HCl to partially depurinate the DNA
2. Soak the gel in dilute NaOH solution so it can cleave at depurinated sites
dsDNA now ssDNA
, cDNA:
isolate mRNA, RT to ds cDNA, choose vector, insert cDNA, create phage or
bacterial library with oligo-dT matrix as backbone
compared to cDNA libraries, genomic libraries are "easy"
1) fragmentation to appropriate size for vector being used.
For lambda = ~20kb
same as cDNA
Topoisomerase relieves torsional stress
Klenow fragment is enzymatic product of DNA poly I from E. coli, retains
polymerase action
Lacks 5'—>3' exonuclease
1. study effects of point mutations found in diseases
2. investigate binding site of proteins
3. determine effects of a deletion within an enzyme
Don't know?
3 of 100
Term
In the figure below, fill in the blanks:
mRNA---->mRNA-DNA hybrid---->ssDNA ---- >dsDNA
Correct
Incorrect
1 of 100
Term
Explain the steps of site directed mutagenesis
Give this one a try later!
plate with bacterial lawn
lay nylon membrane over plate to lift off replica of plaques
membrane acts like Southern blot membrane
Blocking with nonspecific DNA, and using a transfer with a lumescent probe that will
bind and light up sequences of interest
detect with autoradiography
,minimum base pairs required for single primer self-dimerization = 5bp
minimum bp for hairpin: 4bp
consider:
hybridization to other sequences = check via BLAST
secondary structure and Primer interactions
Is it G:C or A:T at 3' end - depends on your application
3' deltaG
Melting temperature differences
4 main types:
1. Ligation-mediated mutagenesis
2. PCR/Primer Extension mutagenesis
3. QuikChange mutagenesis
4. Inverse PCR mutagenesis
STEPS
1. Need plasmid: most replicated by E coli
2. Need ORI, MCS, Ap^r
3. plasmid transformed into E. coli
4. isolate small amount of plasmid DNA
5. screen plasmid DNA for mutation by 1: restriction site or 2: southern
hybridization
6. screen all mutated segments
NEED to DESTROY RESTRICTION SITE
EcoRI cuts at a site, digests, Klenow + dNTPs, T4 DNA ligase to put back
together
, Negative: for PCR, No template. For Ligation, no ligase. For DpnI, undigested
plasmid
Positive: For PCR, Control Plasmid with Primers. For Ligation, plasmid cut with 1
enzyme and ligase. For DpnI, control plasmid amplified with control primers
Don't know?
2 of 100
Term
State 2 ways in which you can increase the transfer efficiency of DNA
to a blot.
Give this one a try later!
1. Soak the gel in dilute HCl to partially depurinate the DNA
2. Soak the gel in dilute NaOH solution so it can cleave at depurinated sites
dsDNA now ssDNA
, cDNA:
isolate mRNA, RT to ds cDNA, choose vector, insert cDNA, create phage or
bacterial library with oligo-dT matrix as backbone
compared to cDNA libraries, genomic libraries are "easy"
1) fragmentation to appropriate size for vector being used.
For lambda = ~20kb
same as cDNA
Topoisomerase relieves torsional stress
Klenow fragment is enzymatic product of DNA poly I from E. coli, retains
polymerase action
Lacks 5'—>3' exonuclease
1. study effects of point mutations found in diseases
2. investigate binding site of proteins
3. determine effects of a deletion within an enzyme
Don't know?
3 of 100
Term
In the figure below, fill in the blanks:
mRNA---->mRNA-DNA hybrid---->ssDNA ---- >dsDNA
