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 � 2. Isolate food/plant DNA �

3. PCR of Plant and �35s or Bt genes�

4. Gel �Electrophoresis �

5. Internet Exploration: ��All About GMOs��

Overview of GMO PCR Lab

GMO+ GMO-

Fall 2011, page 1

!

!

!

!

!

Extension)

Annealing)

Denatura1on)

Add plant or food product

Add Lysis Buffer Add Lysis Buffer

Transfer 400µl of liquid

Grind

Centrifuge

Lab Flow, Parts 1 & 2

Incubate on ice Centrifuge

Part 1: Food Lysis �

Part 2: Removing Impurities�

Fall 2011, page 2

Mix Boil

Add 40µl of NaCl

Add 400µl of Isopropanol Mix

Remove liquid

Transfer 300µl of liquid Centrifuge

Dry DNA pellet Resuspend DNA in TE/RNase

Lab Flow, Part 3

Store on ice

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 200µl 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 800µl 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 for the 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 400µl of the clear liquid

(step 9)

Proteins settle to bottom

Lipids float on top Take liquid

from here

Fall 2011, page 7

Incubation on Ice

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

(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 300µl of the liquid into a new tube and add 400µl 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 200µl (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 200µl TE/RNase

(step 20)

Fall 2011, page 11