Random
What does blue mean on the NFPA Hazard Diamond?
Health Hazard
Define plasmid
Define transformation
transfer of genetic material into prokaryotic cells
Fill in the missing RT-PCR steps
1. Linearization of RNA
2. Chilling period
3.
4. Adding RT
5.
6.
7. adding RNase H
8. degredation of RNA
3. Activation of buffer
5. Retrotranscription (cDNA synthesis)
6. deactivation of retro transcriptase
RT-PCR is quantitative of qualtitative?
Qualitative
What is the A260/280 ratio for DNA?
1.8
What step immediately follows lysing the cells in plasmid purification?
Neutralizing the lysate
Define transfection
transfer of genetic material into animal cells
2 Step RT-PCR method
used when quantity of sample is limited, to store cDNA for later use, to analyze multiple targets
q-PCR
amplify target sequences using cDNA template, primers, fluorophore/probes to quanitfy level of expression
What does a low Ct mean?
High amount of starting material in PCR
Steps of PCR reaction
Denaturation
Annealing
Extension
Most popular ways to transform prokaryotic cells?
Heat shock (chemical transformation) and electroporation
1 Step RT-PCR method
faster and less manipulation of sample, perfect for robotic methods, less pipetting error, cannot store sample, limited in number of target genes
RT-qPCR
similar to qPCR but RNA are directly used and transcripted to cDNA prior to PCR
What is an internal control in qPCR?
reference gene like actin to normalize DNA content
What is external control in qPCR?
Wildtype/cell with no drugs to compare the increase or decrease of expression
Most popular ways to transfect animal cells?
Lipofectamine (lipid based vesicle transfer) and electroporation
What temperature does RNA linearization occur at?
65C for 5 min
When does Syber green bind during qPCR?
Binds after annealing and extension, binds to double stranded DNA
How many times do you repeat PCR cycles?
35-40 times, increase more when unsure what starting amounts were to ensure signal is detected
What is the ratio of ribosomal RNA for prokaryotes and eukaryotes for gel electrophoresis?
Prokaryotes: 23S and 16S
Eukaryotes: 28S and 18S
express transitory transgene in plant tissue to assess the effects before investing in making a stable transgenic line
Absolute quantification
how many DNA have in sample, need to use standard curve to comapre signal to a known reference
Relative quantification
how much sample has increased or decreased compared to the control/wildtype