Lab 2 - Agarose Gel Electrophoresis & Gel Purification

Info from Lab Handout

last week carried out PCR to amplify the

protein-coding region of 1/2 genes that we hypothesize might be involved in transcriptional regulation

these transcription factor proteins (that we amplified) can influence the transcription levels of

certain target genes that they control

WHY did we amplify the gene via PCR - ultimate goal

synthesize and study function of potential transcription factor proteins

before can study after PCR need to

capture the DNA encoding these proteins

what use to capture DNA encoding these proteins

bacterial mini-chromosome called plasmid aka vector aka carrier (of DNA)

plasmids are capable of replicating inside a bacterial cell and once gene is placed into plasmid

your gene gets replicated along with the plasmid DNA

cloning

process of capturing piece of foreign DNA into bacterial plasmid

then replicating the plasmid in bacteria

heterologous protein expression

transcription and translation of a foreign gene within bacterial cells

bacteria can replicate foreign genes of any org once cloned into plasmids and express foreign genes as proteins

purified proteins are also valuable bc we can use them to

generate antibodies

WHY bother express euk proteins in bacteria

1. often hard obtain particular protein directly from org’s own cells, esp if don’t make much of it

2. bacteria cheap, ez to grow

3. we have trick for purifying “our” protein away from bacterial proteins - process much easier/efficient than trying to isolate from org’s own cells

color of liquid that we PCR’d

clear once started and clear once ended

agarose gel electrophoresis

technique used to help visualize presence, size of DNA in sample

electrophoresis

standard technique used for separating molecules based on size and electrical charge

many diff types

later in semester will be using what to separate proteins

different technique SDS-polyacrylamide gel electrophoresis

agarose

porous gel polysacc purified from agar which itself is isolated from seaweed

polymers made of repeating units of disaccs

Agarose polymers can dissolve in buffer at high temps but

they aggregate with e/o to form a solid gel at room temp

as agarose polymers cool, begin to

hydrogen bond with e/o - first double helical structures > aggregate form gel (pore sizes vary with [agarose])

once agarose dissolved by heating it in a buffer solution, melted agarose poured into gel caster. As this solution solidifies as it cools >

makes matrix like stiff jello shape of gel caster

use plastic gel comb to

make small indentations “wells” in gel > hold DNA samples being studied

the gel placed into gel box where it will be submerged in running buffer that allows

generation of electrical current > flows thru gel to separate DNA fragments

how DNA move

DNA backbone phosphate groups carry (—) in slightly basic pH of buffer used,

migrate toward (+) pole during electrophoresis in the apparatus

agarose matrices serve as

molecular sieves

how separate based on size

smaller frags move through pores faster relative to larger molecules, traveling longer distances

difference between 1% agarose vs 3% agarose

can change size of pores, more = smaller

markers/ladders

DNA fragments of known lengths put in separate well compare migration of unknown DNA frags

compare to approximate lengths

DNA fragments are invisible in the gel and

can’t be seen unless they’re subjected to staining

one common method of detection is to stain DNA frags using chemical like ethidium bromide which

slides (intercalates) in btwn base pairs of double helix

gel either submersed in EthBro solution after complete GelElectro or EthBro can be added directly to agarose before casting the gel

when handling ethidium bromide

be careful wear gloves bc suspected carcinogen

if concentration is high enough can visualize by eye but most time visualization requires

illumination using UV light

ethidium bromide molecs emit bright orange fluorescent color

ethidium bromide =/= loading dye, what loading dye

added to fragments prior to electrophoresis has (4)

• Tris-HCl buffer maintain pH

• EDTA inactivates DNA-destroying enzymes by chelating divalent cations

• Glycerol/Ficoll to sink the sample to bottom of well

• 1+ tracking dyes (common bromophenol blue/xylene cyanol) - dyes help color sample to see ez when loading, dyes move through gel at set pace, keeps track roughly how far DNA has migrated thru gel even if can’t see DNA directly

After amplifying piece of DNA of the correct predicted size, next step

cut PCR band out of agarose gel and purify DNA away from agarose > digest DNA with enzyme that will help us clone PCR frag into a vector

cut out slice of agarose with DNA > microfuge tube, next place into special solution with chaotropic salt used to

disrupt Hbonds of gel w/ heating > breaks down agarose to release DNA and keeps gel liquid at room temp

how a chaotropic salt work (like sodium iodide/guanidinium chloride)

helps disrupt intermolecular interactions ex: Hbonds, hydrophobic effects (noncovalent) and remove water from hydrated molecs

DNA fragments now in solution will be captured using silica resin binds to DNA frags

SiO₂ Resin - technique first developed by Vogelstein and Gillespie, 1979

slightly acidic pH of chaotropic salt solution helps DNA interact with silica resin. once captured,

DNA fragments can be washed free from salts, enzymes, and impurities > eluted from silica resin for use in other procedures

will be using kit provided by Qiagen, why these kits often used in molecular bio

provide researcher w all relevant reagents and easy to follow protocol, usually designed to be more foolproof than if u made urself

PROCEDURE 1: Agarose Gel Electrophoresis Step 1

Make 1% agarose solution dissolved in running buffer (0.5X TBE) supplemented with ethidium bromide solution

25 uL of a 10mg/ml EthBrom solution per 500ml gel

TBE (running buffer) is made of and functions

• Tris buffer - keeps solution slightly basic

• Boric acid - provide ions for current

• EDTA - chelates divalent cations used as cofactors by many enzymes including DNAses

Dilute 5/10X stock to 0.5X working conc

PROCEDURE 1: Agarose Gel Electrophoresis Step 2

Heat agarose-buffer solution in microwave until all agarose dissolved into buffer

• hot very quick, can boil over, don’t overheat

• if in bottle, don’t screw on lid tightly can explode!

PROCEDURE 1: Agarose Gel Electrophoresis Step 3

Set up caster and insert clean comb into slots on gel mold

PROCEDURE 1: Agarose Gel Electrophoresis Step 4

Allow heated agarose to cool then pour into gel caster WEAR GLOVES cuz EthBrom

PROCEDURE 1: Agarose Gel Electrophoresis Step 5

When gel completely solidified, remove comb carefully

Place gel into electrophoresis chamber (gel box) and completely submerge gel w buffer

Place wells in appropriate position to run DNA toward positive pole (red)

PROCEDURE 1: Agarose Gel Electrophoresis Step 6

BEFORE loading sample

load molecular size marker into 1 well (1.0 kilobase ladder, has DNA sizes tend increase by 1000 bps)

Load 10 uL of marker into single well of gel

PROCEDURE 1: Agarose Gel Electrophoresis Step 7

in 1,5 mL microfuge tube mix:

• 5uL of 6x loading dye with

• 25uL of PCR rxn

Mix then load into one of the wells of gel - don’t puncture !

PROCEDURE 1: Agarose Gel Electrophoresis Step 8

Once gel loaded replace gel box top (if gel moved, samples can come out of wells)

Ensure all wires connected properly (black-black/neg and red-red/pos)

Turn on power source - typically 100 Volts

PROCEDURE 1: Agarose Gel Electrophoresis Step 9

After gel electrophoresis complete, visualize DNA frags with hand held UV light source/UV box

GOGGLES UV damage eyes

PROCEDURE 1: Agarose Gel Electrophoresis Step 10

After separation and visualization, use sharp remove block agarose w fragment

GOGGLES

> microfuge tube labeled

PROCEDURE 2: Gel Purification Step 1

Normally would weigh but will assume slice is 150 mg to save time

PROCEDURE 2: Gel Purification Step 2

Add

• 3 volumes of QG (yellow solution) for every 1 volume of slice (1mg=1uL)

  • ex: 450uL QC for slice weighing 150 mg

High conc chaotropic salt solution is designed to

help dissolve the agarose to release DNA fragments

Kit uses Guanidine Thiocyanate

Buffer QG also

decreases water-DNA interaction while favoring interaction DNA-silica resin and lowers pH to below 7

(contains buffering agent at about pH 6.6)

PROCEDURE 2: Gel Purification Step 3

Incubate 50-60 deg C

mix several times to make sure agarose slice dissolves completely

PROCEDURE 2: Gel Purification Step 4

Add 1 volume isopropanol = to original slice volume

• ex: 150 mg slice = 150uL isopropanol

Why use isopropanol

Isopropanol affects solubility DNA and helps DNA stick to resin

PROCEDURE 2: Gel Purification Step 5

Label blue/purple spin column and place into 2mL collection tube

Add all of sample but not exceeding 800uL

PROCEDURE 2: Gel Purification Step 6

Centrifuge column+collection tube at max speed 1 min

Discard flow thru

What happens in Gel Purif step 6

DNA frags bind to silica resin in columns as solution flows over the resin

PROCEDURE 2: Gel Purification Step 7

Add 750 uL of wash buffer PE to spin column

Let incubate for 1 min at room temp

Spin again max speed 1 min

What wash buffer do Gel Purif step 7 AND what wash buffer mostly contain

Helps remove salts from buffer QG while allowing DNA to stay bound to silica

Ethanol

PROCEDURE 2: Gel Purification Step 8

Discard filtrate (stuff at bottom collection tube)

Spin column+empty collection tube again (remove residual wash buffer)

DONT SKIP residual traces alcohol will cause downstream problems

PROCEDURE 2: Gel Purification Step 9

Label 1.5 mL microfuge tube

place spin column into tube now used as collection tube

PROCEDURE 2: Gel Purification Step 10

Add 30uL elution buffer (EB) to center of spin column (white resin)

let sit for 1min

After incubate centrifuge column+tube at max speed 1 min

What happen failure to add elution buffer directly into the resin

decreases recovery of DNA

the DNA can bind at

neutral/low pH but not at higher pHs

the elution buffer has a low salt conc and higher pH (8.5) than

buffer used to bind DNA to resin (which has lower pH)

low salt and high pH of EB interfere with

binding of DNA to silica

so DNA washes right off

PROCEDURE 2: Gel Purification Step 11

discard blue/purple spin column

Clean, agarose and salt free DNA now in bottom of collection tube ready for next step (next lab!!!!!)

Buffer QG vs Elution Buffer (EB)

Buffer QG

high-salt buffer, with high [salt] for slightly acidic (6.6 i think) envir.

used to bind DNA to a silica membrane during a gel extraction procedure

• Guanidinium thiocyanate

Buffer EB

low-salt, basic (8.5) buffer used to elute the purified DNA from the membrane by disrupting its binding

• Tris-HCl