Download Genetics Lab - Identification of a Nucleic Acid

Investigative Lab – The Case of the Virulent Virus
Week One: Is the nucleic acid DNA or RNA?
Overview
Each group will be given a nucleic acid sample to analyze over the next few weeks. You
must determine whether the nucleic acid is DNA or RNA, whether it is single-stranded or
double-stranded. Based on this information, you should be able to identify the Virulent
Virus.
The following equipment and reagents will be available for week one, and might be
helpful when designing your experiments.
Bial’s Reagents (tests for riboses)
Dische Reagent (tests for deoxyriboses)
Thermal cycler set to 94°C
Pipets and tips
Distilled water
Gloves
Technique: Reaction of nucleic acids with Bials Reagent.
Background
In the presence of concentrated acid, RNA can be hydrolyzed to a mixture of
purines, ribose sugars, and pyrimidine nucleotides. The ribose sugars the will then
dehydrate to form furfural intermediates that react with orcinol in the presence of ferric
ions to produce a blue-green color (max at 660 nm).
Procedure - Bials Reaction
1) In a 0.2 mL microcentrifuge tube, add the following: 5 g nucleic acid, 60 L
orcinol acid reagent, and 40 L of 6% alcoholic orcinol.
2) Mix each tube, and put in the thermal cycler at 94°C for 20 minutes. Record
your observations.
3) Allow samples to cool completely, then measure their A660 on the
spectrophotometer.
Technique 2: Reaction of DNA with Dische Reagent
Background
In the presence of concentrated acid, DNA is first hydrolyzed to form purines and
pyrimidine nucleotides, then the deoxyribose sugars generated are dehydrated to form hydroxylevulinyl aldehydes which react with diphenylamine to form a blue color (max
at 595 nm).
Procedure - Dische Reaction
1) In a 0.2 mL microcentrifuge tube, add the following: 5.0 g nucleic acid,
sterile distilled water to a total of 30 L, 60 L diphenylamine reagent.
2) Mix and put in the thermal cycler at 94°C for 10 minutes. Observe any color
changes.
3) Allow samples to cool completely, and read the A595. Record and explain your
results.
MATERIALS REQUEST SHEET FOR GENETICS
Lab Day:
Group Members:
Experiment(s) Chosen:
Solutions/Chemicals Needed (include volumes):
Equipment Needed: