Teaching Supplement for Chelex DNA Extraction

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

v Contains photos documenting the key steps in the DNA extraction procedure

vPhotos are numbered according to the BABEC Student & Teacher Guides for ALU, mtDNA, & D1S80 PCR

v Can be used for a pre-lab discussion or during lab for troubleshooting & support

Boil

Lab Flow: Chelex DNA Isolation ProcedureRinse mouth with saline

Resuspend

Mix

Store on ice

Fall 2011, page 1

Pour off liquid

Centrifuge

CentrifugeTransfer saline

Transfer cells into Chelex

Transfer DNAout of Chelex

A microfuge tubehas a total volume of 1.5 milliliters (ml), or 1500 microliters (µl).

1500µl

1000µl

500µl

100µl

Fill the tube with your saline rinse to a volume of 1500µl(step 4)

1500µl

Introduction to microfuge tubes

Fall 2011, page 2

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

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

After centrifuging, look below the hinge for the solid material (pellet)Fall 2011, page 3

After you centrifuge the saline rinse, your cheek cells will settle to the bottom of the tube. This is called a cell pellet.

The cell pellet will be white. Observe your cell pellet. If you do not see one, add more saline rinse and repeat.

The clear liquid above the cell pellet is called the supernatant. It does not contain any cells.

When you place your tube in the centrifuge, have the hinge of the tube facing outward. Then you can find your cell pellet directly underneath the hinge.

Cell pellet side viewCell pellet back view

Supernatant

Cell Pellet

The Cell Pellet

Fall 2011, page 4

The supernatant can be poured out ofthe tube by inverting it.

The cell pellet will stick to the bottom of the tube while you pour.(step 6)

Observe cell pellet stuck to the bottom of the tube while you pour.

If you do not see one, repeat withmore saline rinse.

Decanting the Supernatant

Fall 2011, page 5

There will be about 100µl of supernatant remaining in the tube after you pour. If not, add more saline to bring the volume up to 100 µL.

To resuspend means to dislodge the cell pellet and mix in into the remaining liquid using your pipette.

(step 7)

After resuspension, the solution will be cloudy.

This is because you have concentrated your cells into a smaller volume than what you started with.

100µl

Resuspending the Cell Pellet

Fall 2011, page 6

Chelex is made of very small white beads.They are heavy and settle to the bottom on the tube.

Chelex can’t be pipetted with a P20 or P200 tip. It will shear the beads. Use a P1000.

Chelex beads mixed with your cell suspension(step 9)

Tiny Chelex beads

Chelex

Fall 2011, page 7

After heating and centrifuging, DNA is in the supernatant.

Chelex beads& cell debris

DNA is in thesupernatant

Remove DNA with a pipette(step 13)

Keep the pipette tip near the surface of the liquid so asto not touch the Chelex beads.

Keep pipette tip near the top of the liquid while aspirating

Removing the DNA

Fall 2011, page 8