GENET 390: Topic 3.1 - Detecting Nucleic Acids (gel + PAGE electrophoresis)

We use Gel electrophoresis to separate molecule based on ____?

Size

What are the 4 Ingredients needed to run a electrophoresis Gel?

1. Agarose

2. Sample

3. Buffer

4. Electrolytic cell

Why is agarose the most common type of gel used for DNA?

• Cheap

• Easy

Is agarose a Linear or branched polymer. What repeating unit is it made up of?

Linear polymer of Agarobiose (a Disaccharide made of D-galactose + 3,6-anhydro-L-galactopyrranose)

Why is Agarose EASY TO WORK WITH?

Can be easily melted + repolymerized in a typical microwave

What does Agarose do in the context of Gel electrophoresis?

Creates long polymers that form a MOLECULAR MESH

• act as a sieve to slowdown larger molecules

What 3 things does the velocity of the nucleic acids through the matrix depend on?

1. Charge

2. Mass

3. Shape

In gel electrophoresis we only want to judge mobility based on what?

Mass

Why is it easy to judge DNA based only on mass with gel electrophoresis?

• DNA = uniformly negatively charged

• Linear

How do you manipulate agarose to meet the needs for the size of DNA you need to separate?

Make gels of Different Percentage of agarose

What occurs as you INCREASE the concentration/percentage of agarose used?

Resolve SMALLER fragments of DNA

• smaller pores

What is the Percentage of agarose that is good for day to day use? What size of DNA do you separate with it?

1%

• medium sized (0.5 kb - 1Kb)

Does gel electrophoresis have a max size that it can resolve? If yes what is it?

Yes

• Molecules larger than 50kb cannot be resolved efficiently by standard gel electrophoresis

What property of DNA allows gel electrophoresis to distinguish fragments purely by length?

DNA has a constant Mass/Unit length + constant charge = all fragments move toward the positive electrode at rates determined only by their size.

What shape must the DNA be to be run correctly?

LINEAR

What happens if you run Plasmid or gDNA?

Circular/ Supercoiled

• will run father than their bp would otherwise suggest

What buffers (2) is used for gel electrophoresis?

The agarose monomers are dissolved in Tris-based

• TBE (tris-borate EDTA)

• or TAE (tris-acetate EDTA)

What pH are the buffers for gel electrophoresis?

~ 8.3

WHY is the pH of the buffers for gel electrophoresis what they are?

Want a HIGHER pH

• DNA = stable in neutral - slightly basic environments

Acidic environment = protons will go after the PO4- + disturb the negative charge needed or it may hydrolyze the backbone with acid induced damage

Run to the ____

Run to the red

Nucleic acids are poly-______ therefore will migrate towards the _____ electrode

Poly-anions

Migrate to the POSTIVE Electrode

How much VOLTAGE should be applied?

Depends on how long you want it to take

Other than how long you want the gel to run, What 3 other things affect how much voltage you should use?

1. DNA size

2. Type of Buffer

3. Length of Gel

What is the usual rule of thumb for how much voltage to use depending on gel length?

2.5-10 volts/cm

What happens if you use too much voltage?

• SMEARS

• Gel melts

How does the % of agarose in gel affect the Voltage used?

Lower percent gel = Lower voltage

Why is 0.4% agarose annoying to work with?

Low percentage of agarose = low voltage used

What are the 2 ways to SEE the DNA?

1. Ethidium Bromide (EtBr)

2. SYBR safe

How does EtBr work to visualize DNA?

Fluorescent INTERCALATING AGENT and absorbs UV light + reemits it

Define INTERCALATING agent

Slides between the DNA bases

Why can EtBr intercalate between DNA bases?

Its a PLANAR molecule

What is the most effective wavelength of UV for EtBr fluorescence?

254 nm

Do we use 254 nm for EtBr fluorescence? Why or why not?

No

• It damages DNA

If we don’t use 254 nm, what do we use instead?

300 nm

What should you do in terms of UV exposure if you plan on using the DNA after the Gel?

Minimize the UV light exposure

What Color of light does EtBr reemit after being excited? What wavelength?

ORANGE

• 590 nm

****What are 4 Problems with using EtBr?

1. Intercalating agent (distorts stack) = CARCINOGENIC (cause DNA poly slippage during replication)

2. UV visualizer will burn eyes + skin

3. Extended exposure of EtBr (mutagen) treated DNA with UV light (Mutagen # 2) will cause DNA cleavage

4. Smaller fragments have less fluorescence

What are the 5 advantages of SYBR Safe?

1. Less chemically toxic

2. Fewer disposal issues

3. Better sensitivity than EtBr

4. Low background staining

5. Stains RNA + DNA

What are the 2 disadvantages of SYBR Safe?

1. expensive

2. Stain only useful for One gel

What are the 2 ways to Measure DNA present on gel?

1. Estimating length

2. Estimating Mass

What is used to estimate length of DNA present on gel?

Molecular wt marker/ size standard/ ladder

• Comparing your fragment with the size standard/ladder = estimate length of DNA

What are the 2 common ladders used?

1. Lambda HindIII

2. 1 kb ladder

To what part of the DNA band do you always measure to?

crisp, MIDDLE + BOTTOM of the band

Is the Migrating down the gel uniform?

No

• distance between each bk is not constant down the ladder

When comparing the length migrated to the Standard curve of the ladder to get the size of the DNA on the gel, can you use the same standard curve for different gels? Why or why not?

No

• need to create a standard curve for EACH gel

• Run length differs between gels + voltage + time run

What can you do/use to estimate MASS of DNA on gel?

EtBr Flourescence

Why can you use EtBr fluorescence to estimate mass of DNA?

DNA length is PROPORTIONAL to EtBr Fluorescence

• EtBr Intercalates at a constant rate

Thus you can compare the BRIGHTNESS of EtBr with the Linear DNA ladder fragments to estimate the mass of each Fragment

How often does EtBr intercalate with DNA?

EtBr intercalates every 2 bp

How does Estimating DNA LENGTH + MASS differ?

Length = Size of DNA fragment bp + compare to ladder of known DNA LENGTHS

Mass = quantity how much DNA there + compare to Ladder of known DNA MASSES

What do you need to know about your ladder in order to estimate the mass of your DNA?

The MASS of the ladder

How to actually Estimate mass using ladder?

If your band at same DNA length:

• Glows about as bright as a 100 ng ladder band → it contains roughly 100 ng of DNA.

• Glows half as bright → about 50 ng.

• Twice as bright → about 200 ng.

Mass affects how bright it glows, but longer DNA naturally carries more dye (and therefore appears brighter if equal molecule numbers)

Does Running an RNA gel differ from running a DNA gel? Why or Why not?

Yes

• RNA is DIFFERENT IN STRUCTURE

*****How are RNA different in Structure + what must be done to mitigate that?

RNA loves forming double stranded SECONDARY STRCUTRES + impact the migration

• Secondary structures must be DENATURED before running the gel

******What are the 3 ways to Denature Secondary RNA structures before running a gel?

1. Formamide + Formaldehyde in the loading

2. Heat

3. Use MOPS Buffer

What does Formamide + Formaldehyde do to denature RNA?

It interferes with the H-bonds = holds the double strands apart + keeps it denatured

At what temp + for what amount of time is the RNA heated to denature?

65 for 15 minutes

Why us a MOPS buffer? What does it do?

MOPS = 7 pH = drops the pH = less stable

Are migration rates the dame between denatured RNA + DNA? Can you use the same markers?

NO, use RNA markers

• RNA = ss

• DNA = ds

Can EtBr still be used to stain RNA gels?

Yes

*******What are the types of Buffers used for DNA vs. RNA gels?

DNA = TRIS based

RNA = not tris based MOPS

*******What are the 3 reasons for why RNA gels do not use TRIS based buffers?

1. RNA = unstable at typical pH of tris buffers (greater than 8)

2. Formamide + Formaldehyde form H-bonds with RNA to stabilize ssRNA @ pH 7 and not pH 8

3. Formamide + Formaldehyde preferentially interact with Tris rather than dsDNA

Fill in the blank: Majority of plasmids in vivo are ______ and most are ______ therefore the migration patter is _______ than linear dsDNA

Majority = Circular

Most = Supercoiled

Migration patterns = Much different

*****What are the 3 forms of uncut plasmid DNA?

1. Open circular (OC): Single strand NICK in DNA = Relieves super coiling AKA. RELAXED

2. Covalently closed Circular (CCC) AKA. Supercoiled (SC)

3. Linear: Rare/ never seen in uncut preps

Rank the 3 forms forms of plasmids from fastest runnign to slowest running?

1. CCC/SC super coiled

2. Linear

3. OC (big + gangly)

****What form of uncut plasmid do you want to used on gels? Why?

SC/CCC

• represents intact + Unaltered plasmids

• Natural state in vivo

****What needs to be done in order to confirm that the plasmid is the Right size?

Linearize with RE digest

• migrates according to its true size (can confirm plasmid length matches with what is expected)

What are Gel extractions/Band prep and why you want to do them?

Cutting a DNA band out of an agarose gel after electrophoresis and purifying the DNA from the gel slice.

• You can isolate a specific DNA fragment from a mixture to use in another experiment

What are some examples of what you can do with a gel extraction?

1. Clone + Fragment

2. Label

3. Sequence

*****What are the 4 steps to the Band prep proceedure?

1. Cut out band + Dissolution

2. Separation

3. Wash DNA

4. Elute

What is Dissolution, What exactly is occurring?

Dissolve gel with heat + buffer

What buffer is used in dissolution of DNA and Why?

Tris = stabilize DNA

What is Separation, What exactly is occurring?

Bind DNA to a silica gel membrane

******How is the DNA bound to the silica gel membrane? What salt [ ], pH and buffers are used? WHY?

HIGH salt + pH <7.5 buffer + Guanidine + Ethanol buffer

• Allows binding of DNA to salts in silica matrix

• Guanidine + Ethanol = strip natural salts associated with DNA to allow DNA to bind with salts on silica

What is used to Wash DNA,+ why? what is removed?

High salt + ethanol to remove guanidine + salt but maintain DNA on membrane

****Why do you want to remove guanidine?

Guanidine denatures proteins = can affect down stream applications eg. RE digest

What is used to Elute?

Low salt [ ] buffer

What is most often used to do band prep?

Kit

• QIAGEN 

PAGE electrophoresis: What is used to make the gel? How does it differ from the gel in Gel electrophoresis in terms of what molecules it can seperate?

PAGE = Polyacrylamide

• TIGHER MATRIX = can separate smaller molecules

What are 3 smaller molecules that are separated using PAGE?

1. ssDNA

2. RNA

3. Proteins

Fill in the blank: Acrylamide polymerizes into ____ (linear/branched) chains + then is cross linked with _______

Into LINEAR chains + cross linked with bis-acrylamide

*****What component of polyacrylamide determines the PORE SIZE?

Bis-acrylamide

• degree of crosslinking = determines tightness of the mesh

*****What 2 Terms determine the FINAL PORE SIZE for PAGE?

1. %T: Total acrylamide concentration per volume (CHAIN)

2. %C: Relative Bis concentration of the total acrylamide (CROSSLINKER)

How does Increasing %T and %C each change the pore size?

Increasing T and C both DECREASE the Pore size

(don’t think is very important) What is a Typical PAGE gel % of T and C?

20% T (chain) + 5% C (cross linker)

ssDNA + PAGE: What is the purpose?

DNA sequencing, oligonucleotide purification etc.

• SMALLER fragments + can see much higher resolution — it can separate fragments differing by just one nucleotide.

True or False: ssDNA must be denatured before running on page?

TRUE

WHY must ssDNA be denatured to run on page?

ssDNA can form secondary hairpins + loops

• Denature = migrate based on size (length) alone

*****What are the 3 Ways to denature ssDNA?

1. Urea

2. Heat with Formamide

3. Run gel @ 70

How does UREA denature ssDNA

breaks H bonds

How does Heat with formamide denature ssDNA?

Heat = denature

Formamide blocks H-bonds = holds ds apart

*******True or false: Running gel at 70 is something you can ONLY do with PAGE + not agarose?

TRUE

• Agarose will MELT

What is an example of using ssDNA + PAGE

Manual sequence aka. Sanger sequencing

*****RNA + PAGE: What is the purpose?

Agarose SUCKS at separating ss or smaller than 300 ntd

What gel should you use if RNA is less than 300 vs. more than 300 ntd?

Less = PAGE

More = Agarose

****True or false: RNA needs to be degraded to run through PAGE like with agarose?

True

What 3 things are done to denature RNA for PAGE?

• Heat + 65 for 15 min

• Formamide + Formaldehyde (prevent renaturation)

• Formaldehyde in gel