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Lysis buffer
detergent disrupting plasma membrane releasing genomic information within, adds salt to solution that will assist in DNA precipitation down the line
RNAse A
degrades RNa to prevent RNA contamination in pure DNA sample
Incubation period for DNA purification
denature cellular proteins for removal from sample
Ice during DNA purification
Prevent refolding proteins, favor hydrophobic aggregation for precipitation out of solution
Precipitation buffer
Precipitate and pellet cellular debris as water soluble materials interact with the buffer ions and hydrophobic protein aggregates precipitate
Addition of isopropanol to DNA purification
ISO is less polar than water- replacing the water molecules that normally surround DNA causing it to precipitate out. Polar salts interact with non polar alcohol and precipitate out of solution
Resuspension of DNA after iso addition
As salts precipitate after iso, resuspension of nucleic acids allows for salt removal
Addition of ethanol to DNA purification
Allows DNA to precipitate while keeping salt suspended
Resuspension in 1x TE
TE is Tris EDTA, where Tris: buffer maintaining pH and EDTA: chelating agent that binds Mg, inactivating leftover nucleases that could degrade DNA
Smearing on genomic DNA gel
Presence of partially degraded RNA fragments
Determining concentration: purpose of 260 nm wavelength?
Ring structures of DNA nucleotides absorb light at this wavelength the most strongly
Problem: RNA has great absorbance here as well
Determining concentration: purpose of 280 nm wavelength? What values indicate high purity?
Aromatic structures in amino acids absorb at this wavelength. A260/A280 value is used to determine purity, where 1.7-2.0 indicate high purity
Purpose of a gel electrophoresis for DNA purification
Integrity and length of DNA
Searching for RNA bands/smears
Unexpected DNA contamination or irregular fragmentation
What about an additional concentration wavelength reading at 320?
Measure turbidity: cloudy/opaque character of solution (Should be clear)
What is an open reading frame?
Sequences of DNA that code for a start codon and at least 100 following amino acids before a stop codon. Theoretically represents a gene that codes for a protein
What size is the fragmentation that occurs in ISO DNA precipitation? Is this a concern?
40-50kb, not a concern, we are working with DNA that is only 2-3 kb long
How do we determine the concentration of our gDNA?
A 260 value X dilution factor X 50 ug/mL = concentration of gDNA
What are the steps for PCR?
Initial cycle: Denature at 95 for 2 minutes
30x cycles: Melt at 95 for 20 sec, Anneal at Tm-5 for 20 sec, Extension at 72 for 3 minutes
Final cycle: Extension at 72 for 3 minutes, store in freezer
What is the temperature, duration, and purpose of denaturation?
95 for 2 minutes. Separate complex DNA into one linear strand
What is the temperature, duration, and purpose of Melting?
What is the temperature, duration, and purpose of Annealing?
What is the temperature, duration, and purpose of Extension?
What is Herc II?
Pfu mutant
Why do we use Herc II and not Taq polymerase?
Proofreading & affinity
What is ArchaeMaxx factor? Why do we use it?
Prevent U poisoning from C→U
Why do we use Tris Borate EDTA (TBE) buffer?
How does DNA move thru agarose?
What is the effect of concentration- higher vs lower- of agarose in a gel?
Why do we use ethidium bromide? Why is it a carcinogen?
Purpose of the positive control
Compare Kanmx to WT length
Purpose of neg control, no DNA both primers
Do the primers anneal to each other?
Purpose of neg control,