DNA Extraction and Quantification of Wheat Samples

DNA Extraction and Quantification of Wheat Samples Using ISO Certified Protocol

AQSA ASGHER* and ZEESHAN ARIF WAHLA

*PLANT BREEDING and GENETICS  DEPARTMENT , UNIVERSITY  OF AGRICULTURE FAISALABAD

SEED SCIENCE And TECHNOLOGY DEPARTMENT,   UNIVERSITY  OF AGRICULTURE FAISALABAD

Corresponding Author’s Email:

[email protected]

DNA Extraction and Quantification of Wheat Samples Using ISO Certified Protocol

AIM:

DNA extraction of wheat samples for micro yield trials (MYT).

SAMPLES:

For the extraction purpose, we actually use 50 samples of wheat leaf.

Precautions:

A fume hood is necessary for handling of organic chemicals.

Reagents:

• α_amylase
• chloroform
• 90% ethanol
• CTAB extraction buffer
• Isopropanol
• RNAase
• Liquid nitrogen

EQUIPMEENTS:

• Oven
• Vortex Mixer
• Freezer
• Water bath
• Fume hood
• Centrifuge

Grinding

Grinding is performed in liquid nitrogen.

First take autoclaved pestle and mottle.

Also, make sure liquid nitrogen is present on bench.

Take 3 to 4 leaves of each wheat sample and grind them manually in pestle and mottle.

Pour liquid nitrogen on samples in pestle and grind them with all your force.

Don’t let your sample turned greenish.

Add more liquid nitrogen and grind them until the sample converted into fine powder.

Take sample powdered in microtubes and the rest of leftover sample is taken in a falcon.

Write up the sample number on the tube and falcon.

Now the further procedure will be performed; CTAB based DNA extraction method according to the certified protocol of ISO.

PROTOCOL:

• Take sample in tube.
• 100 to 200 ml sample can be used according to ISO protocol.
• Add 1ml preheated CTAB buffer.
• Add 10µl of RNase.
• Incubate for 30 minutes in water bath at 65Ċ.
• Agitate it 2-3 times while in water bath.
• Centrifuge for 10 minutes at 12000 rpm.
• Transfer the supernatant in a new tube.
• Add 700µl of chloroform in each tube.
• Centrifuge it for 15 minutes at 12000 rpm.
• Add supernatant in a new tube
• Add 480µl isopropanol mix smoothly by gently inverting the tubes.
• Keep the tubes at room temperature for20 minutes.
• Centrifuge for 10 minutes at 12000 rpm.
• Remove the supernatant and retain the pellet.
• Add 90% ethanol
• Then left the pellet for drying.
• Add 100µl D3 water in each tube containing our purified DNA pellet.

While doing all these steps make sure to change Eppendorf tips each time while handling different samples to avoid contamination.

Further we’ll do DNA quantification to measure DNA quality and quantity.

DNA QUANTIFICATION:

DNA quantification is performed in Nano Drop.

PROCEDURE FOR DNA QUANTIFICATION:

• Open the Nano drop software.
• Select material type DNA, RNA, protein or cell culture in “TYPE” option
• Raise the sampling arm pipet 2µl deionized double distilled water onto the lower measurement pedestal, lower the arm and click the “blank run” option. When the measurement is complete raise the sampling arm and wipe out the sample from both the upper and lower pedestal using a dry lint-free laboratory wipe
• After completing blank run, place 2µl of the sample to be quantified on the lower measuring pedestal and click measure and note the concentration of samples.

Example of wheat Sample readings:

Conclusion:

Nano drop reading of wheat DNA samples should be in the range of 1.8-2.0. Above and below readings indicates that the sample is contaminated during the procedure due to improper safety measures.  The reading below 1.8 indicates presence of protein contamination and above 2.0 show the presence of RNA contamination.