GenomicDNAExtractionPhenol|Chloroform

實驗概要

This section provides a general protocol for genomic DNA extraction using phenol and chloroform.

主要試劑

1.       Glycogen (20 μg/μL)

2.       7.5 M NH₄OAc (ammonium acetate)

3.       Ice bucket

4.       Phenol:chloroform:isoamyl alcohol (25:24:1)

5.       100% ethanol

6.       Dry ice or a –80°C freezer

7.       70% ethanol

實驗步驟

Protocol - Phenol | Chloroform Extraction

1. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to  your sample, and vortex or shakeby hand thoroughly for approximately 20  seconds.
2. Centrifuge at room temperature for 5 minutes at 16,000 ×  g. Carefully remove the upper aqueous phase, and transfer the layer to a  fresh tube. Be sure not to carry over any phenol during pipetting.
3. Proceed to Ethanol Precipitation, below.

Protocol - Ethanol Precipitation

1. Add the following reagents to the aqueous phase, in the listed order in above table.
2.  Place the tube at –20°C overnight to precipitate the DNA from the  sample. Note: If you wish to continue with the protocol, place the tube  in dry ice or at –80°C for at least 1 hour.  
3. Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
4. Carefully remove the supernatant without disturbing the cDNA pellet.
5. Add 150 μL of 70% ethanol. Centrifuge the sample at 4°C for 2 minutes at 16,000 × g. Carefully remove the supernatant.
6. Repeat Step 3 once. Remove as much of the remaining ethanol as possible.
7. Dry the cDNA pellet in a SpeedVac? for 2 minutes or at room temperature for 5–10 minutes.
8. Resuspend the cDNA pellet in 300 μL of TEN buffer by pipetting up and down 30–40 times.
9. Centrifuge briefly to collect the sample, and place the tube on ice.