PRE LAB EXPT 5 (DAY 1)

Nucleic acids

• polymers of nucleotides linked by 3’ to 5’ phosphodiester bond

• can be analyzed spectroscopically because of their bases

Nucleotides

• the building blocks of nucleic acids

• each one is composed of a nitrogenous base, pentose sugar backbone, and a phosphate group

Nitrogeneous bases

can be grouped into two as either purine or pyrimidine

Adenine and guanine

are purine bases

Cytosine, thymine, and uracil

are pyrimidine bases

Thymine

• only found in DNA

• replaced by uracil in RNA

Uracil

replaces thymine in RNA

2 types of nucleic acids

• DNA

• RNA

DNA

serves as repository of genes which carry the biological instruction for the synthesis of proteins

RNA

directly involved in protein synthesis

Purines and pyrimidine

absorb light around 260 nm

Aromatic amino acids

• found in protein

• absorb around 280 nm

To estimate the purity of isolated sample

calculate the ratio of the absorbance at 260 nm and at 280 nm (A260/A280)

Ratio of 1.8

a characteristic of a pure DNA sample

Ratio of 0.6

characteristic of a pure protein

Objectives of day 1

• isolate DNA from strawberry or banana samples

• analyze the isolated DNA using dische reaction

• estimate the purity of the isolated DNA sample

Materials students need to bring

• aluminum foil

• ziplock bag/resealable bag

• 2 pcs strawberries (odd numbered groups)

• ice (per class)

• 1 pc banana (even numbered groups)

• cheesecloth

• paperclip

Day 1 reagents

• homogenizing solution

• ice-cold ethanol

Day 2 reagents

• 6M sulfuric acid

• dische reagent

Materials

• (1) filter paper

• (1) graudated cylinder

• (5) test tubes

• (2) 250ml beaker

• (1) funnel

• (1) stirring rod

• (1) watch glass

Equipment

• stove/hot plate

Safety

• wear PPE and lab goggles at all times

• wash hands thoroughly with soap and water before leaving the laboratory

• dispose gloves properly after performing the experiment

Procedure

• For odd numbered groups, pull off any green leaves on the strawberries. For even numbered groups, peel the banana and remove bruised portions. Record the weight of the strawberry/banana samples and a clean and empty test tube will be used for DNA collection later in the experiment

• Put the samples into a plastic bag (preferrably ziplock bags), seal and gently smash for about 2 minutes

• In a separate beaker, place homogenizing solution and place approximately 30-50ml into the plastic bag

  • avoid making too many soap bubbles while smashing

• Place cheesecloth on top of a beaker

• Open the plastic bag and gently pour the contents into the cheesecloth. You can twist the cloth abve the liquid and gently squeeze the remaining liquid into the beaker

• Tilt the beaker at a certain angle and slowly add ice-cold 95% or absolute ethanol down the side of the beaker

  • the amount (volume of ethanol must be twice the amount of the filtrate

• Allow the mixture to stand for 3 minutes. Collect the floating DNA in one direction only using a spooler

  • the spooler can be a clean wire or a paper clip that has a hook shape at one end

• Place the isolated DNA on the pre-weighed test tube and label

• Cover and store the sample until further use

Waste generated: Excess solvent (ethanol)

disposal container: non-halogenated organic waste

Waste generated: Excess strawberries/bananas

disposal container: biodegradable waste bin