ANDC DU/Biology Protocols/DNA Spooling
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Contents |
Isolation of Genomic DNA from plant tissue (Cauliflower) by spooling method
Requirements
Biological
Cauliflower, kept at -70oC for at least 3-4 hrs
Chemicals
SDS extraction Buffer (100ml)
1M Tris, pH 7.210.0 ml
0.5 M EDTA04.0 ml
3 M NaCl16.7 ml
10% SDS17.0 ml
Raise the volume to 100 ml with autoclaved water.
5M potassium acetate
Chloroform
Isoamyl alcohol
Isopropanol or ethanol
Glassware/Plastic ware
All glassware and plastic ware used should be sterilized
- Conical flasks 100 ml
- Petri plates
- Eppendroffs
- Micro tips
- Pipettes
Instruments/ Equipment
- pH meter
- Weighing balance
- Centrifuge
- Micropipettes
- Mortar and pestle
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 homogenization process. If time is used efficiently, the remainder of the lab can be completed within a 70- 90 minute period.
Procedure
- Take the 5 g of frozen sample.
- Grind the tissue completely using mortar and pestle.
- Put the homogenate in preheated (65oC) for 30 minutes.
- Add 5 ml of potassium acetate and invert mix for 5 minutes.
- Add equal volume of (25 ml) chloroform : isoamyl alcohol (24:1)and invert mix for 5 minutes.
- Centrifuge at 3000 rpm for 20 minutes at room temperature.
- Collect the supernatant and repeat the extraction step once more.
- To the final supernatant add equal volume of chilled isopropanol and mix gently by inversion.
- Spool out the DNA by glass rod and dissolve the DNA in 5-10 ml of TE buffer.
Result
Discussion
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.
| Work in progress, expect frequent changes. Help and feedback is welcome. See discussion page. |
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