Estimating DNA concentration by staining with Fast Blast DNA Stain

 A simple method to estimate the concentration of DNA in a sample
 

Safety warnings:  This procedure uses methanol which is toxic and flammable.  Glove must be worn whenever handling methanol

Materials Needed

100X BioRad Fast Blast DNA stain

PVDF membrane

DNA concentration standards

distilled water

Procedure

Before beginning the assay, practice spotting 1 µl of water onto piece of used PVDFmembrane.

ALWAYS WEAR CLEAN GLOVES WHEN HANDLING THE PVDF (membrane)

1.  Prepare the membrane.

With a soft lead pencil, draw a series of 1 cm by 1 cm boxes, one for each sample and for each of the three standards.

“Wet” the membrane by dipping it in methanol and leaving it submerged until it is uniformly darkened.

Transfer the membrane to a dish of distilled water and allow it to soak until it no longer repels water

2.  Spot the DNA onto the membrane.

The standards will be provided to you by your instructor.  The samples are the samples of DNA that you purified from the plant tissue.

For each standard or sample to be tested, carefully pipette 1 microliter into the center of the box.  If done properly, it will form a bead at the spot where you pipetted it.  This bead will slowly shrink as the liquid soaks into the membrane.

3.  Allow the membrane to dry for 5 minutes.

4.  Repeat the “wetting” process from step 1.

5.  Place the membrane in a dish with enough 100X Fast Blast DNA stain to cover. Shake for 5 minutes.

6.  Transfer the membrane to a dish of distilled water, and shake until you no longer see blue color washing off the membrane.  You may need to change the water while doing this.

7.  Estimate the concentration of DNA in the sample by comparing the intensity of the blue dot from the sample with the intensity of the blue dots of the standards.  The amount of DNA in the standards are 0 ng/µl, 10 ng/µl, and 100 ng/µl.

Purification of DNA from Brassica rapa Tissue.
Collect Tissue for DNA: http://www.youtube.com/watch?v=UYUmWeIaWpQ

1. Place a piece of leaf tissue of approximately 1 cm2 in a mortar and pestle. Add 600 µl of Lysis Buffer and carefully* grind the tissue until liquefied.
Video: http://www.youtube.com/watch?v=RNFT-VM05V4
* Note: When grinding, be very careful not to introduce froth into the homogenate as this will make it very difficult to collect it for the next step. Grind with smooth motions, bringing the pestle across the entire bottom of the mortar before lifting up. The lysis buffer contains detergent so it is easy for it to get frothy.

2. Collect 600 ul of homogenate and place it in a labeled 1.5 ml microtube. If you don’t recover 600 ul of homogenate, add more Lysis Buffer to the Mortar and pestle, grind briefly, collect liquid and add to the homogenate in the tube until the total recovered volume equals 600 ul.

3. Perform an organic extraction:

• a. In the fume hood, add 600 ul chloroform and close the cap tightly. While holding the cap closed, shake vigorously to form an emulsion.   Video: http://www.youtube.com/watch?v=urmyFrIpp2Q
• b. Incubate at room temperature for 5 minutes. During this incubation, mix periodically to keep the mixture emulsified.
• c. Centrifuge at 12,000 x g for 5 min. at room temperature. After centrifugation, you should see two distinct phases and a layer of solid material between them. The upper liquid phase is the aqueous phase and contains the DNA.
• d. Collect the aqueous (top) phase without disturbing the material at the interface. Try to collect the entire aqueous phase. (The DNA is in the aqueous phase.)   Video: http://www.youtube.com/watch?v=7W3zEnupemk

4. Use your pipettor to estimate the volume of what you recovered in Step 5. To this, add an equal volume of isopropanol at -20 degrees C. Mix by inverting several times.

5. Incubate 5 minutes at room temperature.

6. Place tubes in the centrifuge with the hinge of each tube toward the outside of the rotor and centrifuge at 5,000 x g for 5 min. You should see a small, but visible pellet at the bottom. By placing the hinge of the tube to the outside during centrifugation, you can figure out where to look for the pellet. (Think of how the tube is positioned in the rotor and thus where the pellet should form.

7. Remove and discard the supernatant. (The DNA is in the pellet.)

8. Wash the pellet with 70% ethanol.

• a. Add 500 ul of 70% ethanol to each pellet.
• b. Vortex to dislodge pellet. Don’t worry; the DNA will not redissolve in 70% ethanol.
• c. Centrifuge 5,000 x g for 5 min. Remove supernatant
• d. Centrifuge at 5,000 x g for 20 seconds to get all liquid to the bottom of the tube and remove all liquid with a micropipette.

9. Air dry the pellet by leaving the tube open on the bench top. This usually takes less than 10 minutes. However, the criteria is dryness, not time.

10. Redissolve the DNA that is in the pellet by adding 40 ul of TE buffer.

11. Incubate at 55 degrees for 15 min. During this time, mix every 5 minutes.

12. Centrifuge at 10,000 x g for 5 minutes. Collect all of the supernatant and transfer to a new labeled 0.5 ml microtube. Discard the pellet

Store the DNA in the refrigerator.

About this Blog

Human Genetics with Mustard
Doug Wendell and Dawn Pickard, co-PIs
Oakland University, Rochester, Michigan

The goal of our project is to develop DNA markers for rapid-cycling Brassica rapa (aka Wisconsin Fast Plants) and educational materials that use these markers.  Our target is college and advanced high school educators who can use our materials to teach key principles of genetics and the scientific process in general.

As we develop DNA markers and lab procedures we will post the here for all to try.

The purpose of this blog is to share information on the procedures and DNA markers that we develop for rapid cycling Brassica rapa (Fast Plants type).  We will be posting protocols and data on the markers  as we develop them.  The intention is that this be interactive so we encourage users to post comments.  Did the materials work?  What steps did your students have problems with?  What parts do you like?  Do you have suggestions for improvement?

Please also check out our YouTube channel:  http://www.youtube.com/user/HumanGeneticsMustard


Funding
This work was made possible by:
A pilot project grant from the Oakland University Teaching and Learning Committee
Educational Materials Development project grant from the National Science Foundation (grant # 0340910)
An ARRA Award from the National Institutes of Health (grant # 5 RC1 RR030293-02)