Independent Learning Flashcards
How is DNA generated in vitro?
PCR
How are nucleic acids isolated and purified?
- lyse cells
- pellet insoluble material
- use chemicals to isolate nucleic acids from proteins and lipids, or use resin with affinity for phosphate to separate these cell components
- elute purified nucleid acid with ethanol
- analyze for quantity and purity
How is DNA separated from RNA?
- size selection (RNA is smaller that gDNA)
- DNAses and RNAses
- post-transcriptional modifications to RNA may make them distinguishable
How can you preferentially purify mRNA?
use oligo-T primers on resin
How can nucleic acids be identified and quantified?
spectrophotometry and electrophoresis
What is the maximum absorbance of nucleic acids?
260 (in the UV)
What is Beer’s law?
Used to determine concentration based on absorbance
A = epsilon x c x l
what is epsilon in beer’s law?
it is the molar extinction coefficient and varies from substance to substance
What wavelength is absorbed by proteins?
280 nm (in the UV)
What is a pure 260/280 DNA/protein ratio?
1.8. Protein contamination shows lower than this
What is a pure 260/280 ratio for RNA/protein?
- Protein contamination shows lower than this
What would the 260/280 be for 100% protein?
0.57
How can nucleic acids be identified?
incorporation of ethidium bromide, fluorescence, radioactivity
What performs random 32P RNA labelling?
T7 polymerase
What are the specifics for southern blots?
- restriction digest of large DNA
- alkali denatures DNA and gets rid of RNA
What are the specifics for northern blots?
- extraction of RNA over dT resin
- negative RNA transferred to positive nylon membrane, fixed by UV crosslinking and heat
What is a drawback of northern and southern blotting?
You can only see what you probe for, so you have no idea what else is going on in the cell
Benefits of radioactive tracer
high sensitivity
Drawbacks of radioactive tracer
dangerous because mutagenic short half life
Benefits of non-radioactive tracer
dafety and long life
Drawbacks of non-radioactive tracer
low sensitivity
Internal vs external phosphate labelling
internal is alpha, external is gamma
Which enzyme does targeted external DNA 32P labelling
T4 kinase
Describe DNA labelling by nick translation
- DNAse create nick in one DNA strand to remove a sequence
- DNA polymerase synthesizes a new strand starting at the 3’ end of nicked strand
- DNA polymerase exonuclease activity removes nucleotides from 5’ end
Describe DNA labelling by random priming
- DNA denaturation and primer hybridization
- klenow polymerase synthesizes DNA and adds alpha32P dNTPs
How do you generate RNA probes?
- start with cDNA in a vector with T7 or SP6 promotor (cDNA library)
- linearize plasmid with restriction enzyme
- transcription by T7 polymerase incorporates alpha32P NTPs to make a probe
Name two non radioactive tracers
- fluorochromes (direct labelling)
- digoxigenin or biotin (indirect labelling)
Describe how fluorescent tracers work
Probes or sequences are made from nucleotides that are linked to fluorochromes and can be detected with fluorescent microscope
Describe Digoxigenin- conjugated nucleotides
An example of chemiluminescence
- DNA probe is labelled with DIG
- DIG is detected by an antibody which is conjugated to alkaline phosphatase
- addition of enzyme substrate causes a light to be emitted
