ANDC DU/Biology Protocols/Plasmid DNA

= Isolation of Plasmid DNA from E. coli. =

Requirements
Chemicals

A. Luria Broth medium:

Tryptone             1.0g

Yeast extract       0.5g

Nacl                    0.5g

Glucose               0.1g

Dissolve the components in 75ml of distilled water, adjust the pH to 7.6 and finally make the volume to 100ml. Sterilize by autoclaving

B. GTE mix:

50mM Glucose

50mM Tris

10mM EDTA

Sterilize by autoclaving

C. 1M NaOH

Do not autoclave

D. 10% SDS pH 7.2:

Dissolve 10g of SDS in 80 ml of distilled water adjust pH to 7.2. Raise the volume to 100ml.

Do not Autoclave.

E. Mix II

0.2 M NaOH                 2.0 ml

1% SDS                           1.0 ml

DW                                 7.0 ml

F. 3M Potassium acetate pH 4.8

Potassium acetate            29.4g

DW                                 25 ml

Acetic acid                      30 ml

Adjust pH to 4.8 with acetic acid

Raise volume to 100 ml.

Glassware/Plastic ware

All glassware and plastic ware used should be sterilized

·        Conical flasks 100 ml

·        Petri plates

·        Eppendroffs

·        Micro tips

·        Pipettes

Instruments/ equipment

·        Orbital shaker

·        pH meter

·        Weighing balance

·        Centrifuge

·        Micropipettes

Time Required

This lab generally requires more than one lab period. It is suggested that the solutions should be prepared in advance and stored in refrigerator, and have students begin the procedure with the centrifuging process. If time is used efficiently, the remainder of the lab can be completed within a 70-minute period.

Procedure
·       Pick a single colony of E.coli (containing plasmid) from a petriplate and inoculate into 50ml of LB medium in 250 ml conical flask containing 50 µg/ml ampicillin. Incubate the flask at 37 degrees at 250 rpm in a shaker incubator.

·       Take 1.5 ml of overnight grown culture harbouring plasmid DNA in microfuge tube.

·       Centrifuge at 8000 rpm for 5 minutes at room temperature.

·       Add 200µl of GTE mix and resuspend the bacterial pellet. Incubate for 5 minutes at room temperature.

·       Add 400 µl of mix II. Mix well and keep on ice for 5 minutes.

·       Add 200µl of 3M potassium acetate. Invert mix.

·       Centrifuge at 10,000 rpm for 10 minutes. Take the supernatant in a fresh microfuge tube.

·       Add equal volume of isopropanol.

·       Keep at room temperature for 5 minutes.

·       Centrifuge at 10,000 rpm for 20 minutes

·       Decant and dry.

·       Dissolve in 50µl of DW.

·       Load on gel and view the gel in UV.

Precautions
·        Use caution when operating the centrifuge.

·        Dispose of used reagents according to local ordinances.

·        Wear latex gloves and goggles during the procedure.

·        The laboratory surfaces should be very clean during all procedures used in this activity.

·        Use thoroughly clean instruments and glassware. Rinse all equipment with isopropyl alcohol or acetone.

·        Ethanol is highly flammable; use caution.