GMO Teaching Supplement for DNA Extraction

A photo-guide with useful pointers & tips for a successful DNA extraction

Contains photos documenting the key

steps in the DNA extraction procedure

Photos are numbered according to the

BABEC GMO Student & Teacher Guides

Can be used for a pre-lab discussion or

during lab for troubleshooting & support

1. Collect food or plant product 1. Collect food or plant product 2. Isolate food/plant DNA2. Isolate food/plant DNA

3. PCR of Plant and 3. PCR of Plant and 35s or Bt genes35s or Bt genes

4. Gel 4. Gel ElectrophoresisElectrophoresis

5. Internet Exploration:5. Internet Exploration:““All About GMOsAll About GMOs””

Overview of GMO PCR Lab

GMO+ GMO-

Fall 2011, page 1

Add plant or food product

Add Lysis Buffer Add Lysis Buffer

Transfer 400l of liquid

Grind

Centrifuge

Lab Flow, Parts 1 & 2

Incubate on ice Centrifuge

Part 1: Food Lysis Part 1: Food Lysis

Part 2: Removing ImpuritiesPart 2: Removing Impurities

Fall 2011, page 2

Mix Boil

Add 40l of NaCl

Add 400l ofIsopropanol Mix

Remove liquid

Transfer 300l of liquid Centrifuge

Dry DNA pelletResuspend DNA in TE/RNase

Lab Flow, Part 3

Store on ice

Part 3: Isolating the DNA Part 3: Isolating the DNA

Fall 2011, page 3

Use a Small Amount of Food Material

The portion of food/plant material should sit about halfway to the 0.1ml mark

(step 2)

The correct amount of corn meal

0.1ml mark

Fall 2011, page 4

Food Maceration

Add 200l Lysis Buffer to food material

(step 3)

Crush with micropestle (step 4)

Twist and grind with an up & down motion

Keep food debris between pestle and tube wall

If necessary, remove stuck portion from bottom of tube with pipette tip

Add 800l Lysis Buffer to crushed material

(step 5)

Solid material may still remain,but liquid should become cloudy

Fall 2011, page 5

Ideal Centrifuging Method

Orient the hinge of the tube to point outward and away from the middle of the centrifuge.This allows for solid material to settle to the bottom of the tube directly below the hinge.

Hinge of tube points out

below hinge side view

After centrifuging,look below the hinge forthe solid material (pellet)

All spins should be performed at 10,000 x g,which is about 10,000 rpm depending on the centrifuge

If you have a less powerful centrifuge, spin longer than 5 minutes

Fall 2011, page 6

Removal of Food Lysate

Lysate is the solution produced when cells are destroyed.You want to take the clear liquid, which contains the DNA, not the insect debris.

After centrifuging, there may be food debris on bottom of the tube, and possibly on the top

Place your pipette tip between the two layers and take 400l of the clear liquid

(step 9)

Proteinssettle to bottom

Lipids float on top

Take liquidfrom here

Fall 2011, page 7

Incubation on Ice

Food lysate (400l)with NaCl added (40l)

(step 10)

After sitting on ice, the NaCl solution may become cloudy

because a precipitate has formed

The NaCl precipitates the SDS in the lysis buffer. SDS can degrade DNA so you want to remove it.

ice

Fall 2011, page 8

Isolating the DNA

After you centrifuge, the solid material settles to the bottom.

SDS; discard

DNA is in the liquid

Transfer 300l of the liquid into a newtube and add 400l isopropanol

(step 13 & 14)

After the SDS precipitates out, the DNA is in the supernatant, the liquid portion.

Fall 2011, page 9

Drying the DNA Pellet

After the final centrifuge spin, the DNA is now in the bottom of the tube because it precipitates with isopropanol

Small DNA pellet Large DNA pellet

Pour off the liquid& dry the pellet(steps 16 & 17)

No visible DNA pellet

You may or may not see a pellet.DNA is still there even if you can’t see anything.

Here are some examples of what you may see:

Your DNA is here,directly under the hinge

Fall 2011, page 10

Resuspending the DNA

If DNA is cloudy or if there is visible debris, add another 200l (step 21)

Depending on how large your pellet was, your DNA may be clear or cloudy.

Clear DNA Cloudy DNA DNA with debris

Excessive food debris can inhibit the PCR and it helps to dilute it out.

You should still have plenty of DNA.

DNA is resuspended in 200l TE/RNase

(step 20)

Fall 2011, page 11