ISOLATION OF DNA FROM ONIONS

植物 DNA的粗提

Aim

• To isolate nucleic acids from onion tissue.

Note: nucleic acids prepared in this way will not be very pure

Equipment and materials

• Water bath, set at 60℃• Ice in a jug• onion,• Washing-up liquid• Table salt• Protease enzyme• Ice cold ethanol

Principle

• Household detergent is used to degrade both the cell membranes and those surrounding the nuclei.

• Cell fragments are separated by filtration

• The nucleic acids and soluble proteins remain

• Enzyme is used to degrade the proteins• Nucleic acid is precipitated into ice cold

ethanol

Procedure• Add the 3g table salt to the 10cm3 washing-up

liquid

• Chop the onion into small pieces roughly 5mm x 5mm

• Add the chopped onion to a beaker with the salty washing-up liquid solution

• Put the beaker in a water bath at 60℃ for exactly 15 minutes

• Cool the mixture by standing the beaker in an ice water bath for 5 min, stirring frequently

• Filter the mixture into a second beaker. Ensure that the foam on top of the liquid does not c

ontaminate the filtrate

• Add 2-3 drops of protease to about 10 cm3 of the onion tissue extract and mix well

• Pour ice cold ethanol carefully down the side of the onion extract

• Nucleic acids will precipitate at the interface and float into the upper (ethanol)layer

Take some nucleic acids and transfer into TE buffer

Incubate in 60℃ water-bath for 5-10min

Take 10 uL and add 3 uL 6X Load buffer, mix

Load to a 0.6% agarose gel, running the gel under the 120 volts for 30min

Detect DNA under UV light

assignment

• Describe what you observed eventually

discussion