MCB reagents memorization

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 of 10 X 50 ug/mL = concentration of gDNA in ug/mL

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?

95 for 20 sec, break H bonds between bases

What is the temperature, duration, and purpose of Annealing?

ddd

What is the temperature, duration, and purpose of Extension?

72 C, 3 min, DNA synthesis by Herc II

What is Herc II?

Herc II is a fusion between the Pfu DNA Polymerase from P. Furiosus- a DNA polymerase with exonuclease proof reading activity- and a high affinity double stranded DNA binding domain. This combines both high fidelity and high affinity for the PCR reaction

Why do we use Herc II and not Taq polymerase?

We use Herc II due to its high fidelity & affinity to our mutant DNA, as there is no way of knowing if our reaction works IF our reaction works if KAN-MX is mutated

What is ArchaeMaxx factor? Why do we use it?

Cytosine spontaneously deaminates into Uracil, and dUTP nucleotides make Pfu proofreading inefficient. ArchaeMaxx breaks down dUTP to prevent poisoning

Why do we use Tris Borate EDTA (TBE) buffer?

Tris - a buffer that maintains pH

Borate - provides ions to create an electric current in the gel for DNA to “run to red”

EDTA - Sequester Mg from possible nucleases, prevent DNA breakdown

How does DNA move thru agarose?

DNA molecules are negatively charged and migrate to the positive electron. Smaller molecules are able to travel farther as they can slip through the pores of the gel.

What is the effect of concentration- higher vs lower- of agarose in a gel?

Lower concentration = higher resolution of large DNA molecules as the agarose pores will be larger. Causes shorter DNA molecules to move faster/closer together/lower resolution

Higher concentration = higher resolution of small DNA molecules as the agarose pores will be smaller. Causes longer DNA molecules to move much slower/closer to together

Why do we use ethidium bromide? Why is it a carcinogen?

We use ethidium bromide as it intercalates with the DNA base pairs due to its flat, planar structure. The aromatic rings that make it planar are able to fluoresce as their pi electrons are excitable. It is a carcinogen as it can intercalate with our own DNA structures.

Purpose of the positive control

Used to compare the WT length to our mutant length w KANMX knockout

Purpose of neg control, no DNA both primers

Do the primers anneal to each other?

Purpose of neg control, DNA and just one primer (FWD or REV)

Looking for nonspecific binding

What happens if the ion concentration of the gel buffer is too high? What about too low?

Too low - minimal migration, Too high: melt the gel