GENET 390: Topic 2.1 + 2.2 - Nucleic Acid purification + mini prep

****What are the 4 steps of purifying DNA?

1. Precipitation

2. Centrifugation

3. Wash + air dry

4. Resuspend/ dissolve in substrate

****What occurs to the DNA during Precipitation?

Remove SHELL OF HYDRATION = form DNA salt

*****What are the 2 things used to Precipitate DNA?

1. Alcohol

2. Salt/Ion

****What are the 2 Choices of Alcohol?

1. Ethanol

2. Isopropanol

****What are the 2 Choices of Salt/Ion?

1. Sodium Chloride

2. Sodium Acetate

****Of the 2 alcohols, which is more hydroscopic? What does hydroscopic mean?

Isopropanol

• Hydroscopic = ability to remove water

= Need less Isopropanol + easy to use

*****What does the alcohol do + what does the salt do in the process of precipitation? How does it result in precipitation

Alcohol = Removes shell of hydration

Salt replaces where H2O was

• salt provides positive ions (cations) that neutralize the negative charges, reducing solubility → the nucleic acids can clump together and precipitate.

Nucleic acid = forms a salt (negative phosphate + positive ion)

• Preferred association over with water

Less hydrophilic = insoluble = precipitation

*****What is one thing to consider in nucleic acid preparation?

Other material present in initial solution can precipitate + contaminate nucleic acid preparation

****What 3 things will affect the final precipitation results?

1. Source of DNA

2. Purity of DNA required

3. Volume of source material

*******What is LiCl precipitation used for?

selective precipitation of RNA

• Not strong enough to ppt DNA = can act as Differential prep

• Remove DNA contaminants

*****What are 2 cons of LiCl precipitation?

1. Doesn’t work on RNA less than 300 ntd long

2. May inhibit enzyme in down stream applications

***LiCl can be used to precipitate RNA + remove any DNA contaminants. Are we ever worried about RNA contamination in DNA? Why or Why not?

NO

• RNA will self-destruct (very fragile + unstable)

****What is the ROUTINE salt used for DNA isolation?

Sodium acetate

****In what context do we use NaCl as a salt for precipitation?

Isolating DNA with SDS (detergent)

***What is Ammonium acetate used for ?

Salt for DNA precipitation, removes the dNTPs

• selectively help separate DNA from RNA.

****What do we wash the centrifuged DNA pellet with? What does the washing do?

70% ethanol

• washing help dissolve any precipitates salt (help unsalt DNA to resuspend later in solution)

Fill in the blank: after washing the DNA pellet must be ____ before resuspending

Must be dry

• need to air dry

*****What are the 2 solutions that DNA can be resuspended in?

1. TE buffer 

2. Water

****Why can we resuspend in water?

DNA will not denature because of LOW IONIC STRENGTH

• DNA = stable

  • for decades if frozen

*****Why do we resuspend in TE?

More stable for longer

*****What are the 2 things that make up TE buffer?

1. Tris = Buffer

2. EDTA = ingredient that really matters

*****Why is TE more stable for longer?

EDTA

• chelated Mg 2+ = inhibits NUCLEASE ACTIVITY

*****What are 2 cons of working with TE?

1. Harder to work with

2. Inhibits Restriction enzyme digests needed for cloning

• need to rinse TE off before cloning

True or false: RNA is more stable inside the cells than DNA?

True

• but only inside the cells

2 reasons why RNA is less stable outside cells?

1. RNases = everywhere

2. 2’ OH = Will spontaneously degrade outside cell (can CYCLIZE)

*****The spontaneous degradation is even more likely with increasing ____ availability

Mg2+ (DIVALENT)

Charge neutralization

• The RNA backbone has negative charges from phosphates.

• Mg²⁺ shields those charges, making it easier for the 2′-OH to get close to the phosphate and attack.

*****What are the ways to Prevent endogenous + Exogenous RNase? (4 ways total)

Exogenous

• wear glove

• Treat glass at 350 degrees overnight

• Autoclave H2O with DEPC (destroys all biological material including RNases)

Endogenous

• Denature RNases

****How to Denature RNases?

1. Lyse cells (Detergent: SDS or triton)

2. add Organic extraction chemical (phenol)

= creates Mixture of chemicals that break H-Bonds of Carbon in PROTEINS

= Denature proteins (RNase = protein + RNA component)

*****How to separate DNA from RNA?

Acidic phenol/chloroform solvent

• separate aq. + organic phase

*****In which layer is RNA + In which layer is DNA?

DNA + Protein = ORGANIC LAYER

RNA = Aq. layer

*****What are the steps of Acidic Phenol/Chloroform solvent?

*****What is the importance of the Acid in acidic phenol/chloroform solvent?

Low pH = crucial for DNA to solubilize in ORGANIC LAYER

DNA = denatured in acidic conditions

Why do we like phenol/chloroform?

Gentle

HIGH YEILDS

What is another method for isolating DNA from RNA?

Kit

The vast majority of RNA = what type of RNA?

rRNA

*****How to isolate eukaryotic mRNA?

Oligo T affinity chromatography

****What are the steps to Oligo T affinity chromatography?

1. Chromatography column coated in poly-T oligomers

2. Run RNA prep through column (mRNA will bind to poly T bc poly A tail)

3. Wash away unbound material

4. Wash off bound mRNA with High salt

****Why wash of bound mRNA with high salt?

Ions disrupt H-Bond of base pairing between poly A tail + Oligo T

*****What are the Desire Results of Isolating mRNA + Why?

Smear

• no discrete bands bc all mRNA sizes are represented

****What do you not want to see in gel with mRNA isolation? Why?

Smear at bottom

• All mRNA = degraded

***How to test yield? (2 ways)

Spectrophotometric analysis

• RNA = 1 OD260nm = 40 micrograms/microL

qPCR (for specific transcripts)

*****What are the 7 steps to MINI PREP? Mini prep is on the FINAL

1. Solution 1 = Buffer

2. Solution 2 = Denature

3. Solution 3 = Put back together

4. Centrifuge

5. Add Alcohol

6. Centrifuge

7. Resuspend

****What does Solution 1 contain + what does it do?

Tris, EDTA + Glucose

• Stabilize + resuspend culture

*****Specifically what does GLUCOSE + EDTA do?

Glucose = maintain osmotic pressure, don’t want to lyse cells yet

EDTA = Chelates Mg2+ = destabilize bacterial outer membrane

*****What does Solution 2 contain?

NaOH + SDS

****What does NaOH + SDS do in solution 2?

NaOH = Base = denatures DNA

SDS = detergent = disrupt membrane

****What does solution 3 contain?

Potassium Acetate + Acetic Acid

****What do Potassium acetate + Acetic acid do in solution 3?

Neutralize base = Renature DNA

• DIFFERENTIAL STEP

*****Why is Solution 3 the Differential step in miniprep?

DNA strands renature poorly bc its so large + has low supercoiling = remain big + bulky mess

Plasmid = Reanneals easily

****During the Centrifuge step of mini prep, What ends up in supernatant + what ends up in pellet + WHY?

Supernatant = Plasmid

Pellet = gDNA

• bc pellet is small and remains soluble in solution?

****What are 2 possible additional steps that can be done to the supernatant after centrifuge to obtain more pure plasmid?

1. Add RNase

2. Phenol/choroform

****What do Adding RNase + Phenol/chloroform each do?

1. RNase = removes any contaminating RNA

2. Phenol/chloroform = removes any contaminating PROTEINS

****What does Adding alcohol after centrifuge do? How does it work?

Precipitate plasmid DNA from supernatant

• Removes hydration shell + form salt

****Why doesn’t precipitation of mini prep require salt? like isolation of nucleic acids?

There lots already from all the solutions

******What does the 2nd round of centrifuge do?

Concentrate plasmid

****In the last step. What is the plasmid resuspended in? (2 options)

TE or ddH2O

******Pros vs. Cons: Kit vs. traditional miniprep

Pros: Kit = quick + clean

Cons: Kit = lower yields

******Similarities vs. Differences: Kit vs. Traditional miniprep

Similarities: Both have Alkaline lysis

Diff: How DNA = concentrated

******What is Alkaline lysis?

Solutions 1-3

*****How do Kit + traditional DIFFER in how DNA = concentrated for mini prep?

Kit = Don’t use Alcohol

• Instead DNA binds to Silica-gel membrane then washed off with buffer

****How does the silica gel work? WHY DOES DNA bind to silica gel?

1. Add Guanidium = Disrupt DNA interaction with Water

2. Salt wash = Bridges DNA + Silica

• Silica = negative charged DNA = Positive charged due to salt wash 

*****Why does plasmid DNA come off the silica gel?

Remove Salt

• Wash with Low salt Buffer @ a HIGH pH 8.5

Removing salt = charge repulsion between negative silica and negative DNA

****Phenol/Chloroform. When do we use it? (2 scenarios)

• Isolating A LOT of DNA

• Remove PROTEIN Contaminants

EXTRACTING DNA/RNA from Proteins

*****How does Phenol/Chloroform work? What ends up in what layer

DNA = very polar = remains in Aq solution

Organic layer = Phenol + other debris (proteins)

*****BUT WAIT! I thought phenol/Chloroform separates RNA from DNA?

YES but that is for ACIDIC phenol/chloroform

*****How does the acidic nature make a difference?

DNA will dissolve in organic layer if phenol is acidic = used to separate DNA + RNA earlier