Preparation of Genomic DNA
- Suspend bacteria cells in 8 ml of [0.7 M NaCl, 0.1 M EDTA (pH 8.0)] in 50 ml conical tube.
- Add 1 ml of 10 mg/ml lysozyme in PBS.
- Add 30 ul of 100 mg/ml labiase.
- Incubate mixture in 37oC water bath for 1 hour.
- Add 1 ml of [0.5 M tris-Cl (pH 7.5), 5% SDS].
- Add 25 ul of 10 mg/ml protease K.
- Incubate mixture at 60oC for 1 hour.
- Extraction of DNA with phenol.
- Extraction of DNA with phenol-chloroform.
- Extraction of DNA with chloroform-isoamyl alcohol (24:1).
- Precipitation of DNA with cold ethanol.
- Wash precipitated DNA with 70% ethanol.
- Dissolve DNA in 600 ul TE buffer with 1ul of 10 mg/ml RNaseA.
- Incubate DNA at 37oC for 30 min.
- Add 5 ul of 10 mg/ml protease K.
- Incubate DNA at 60oC for 1 hour.
- Extraction of DNA with phenol.
- Extraction of DNA with phenol-chloroform.
- Extraction of DNA with chloroform-isoamyl alcohol (24:1).
- Add 100 ul of 3 M sodium acetate (pH5.5).
- Add 1.2 ml cold ethanol.
- Wash precipitated DNA with 70% ethanol.
- Redissolve DNA in 200 ul TE buffer.
- Dilute 2 ul of DNA into 400 ul dH2O and analyze OD260.
Reconstitution of DNA
- In the case you received plastic vials contains precipitated DNA with ethanol.
- A precipitant might stick to the lid or wall during transportation.
- Before opening the tube, please spin down a minute or two at 5,000 g (~7,000 rpm, table top microfuge).
- Please remove ethanol with pipet (DO NOT tooch precipitate!)
- Immediately after removal of ethanol resuspend the DNA in an appropriate amount of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA), and dissolve the DNA pellet by gently tapping with fingers (DO NOT shake vigorously! A little amount of ethanol won’t affect to many applications).
- If the DNA is difficult to solve, leave the vial at 4 degreeC overnight to allow the DNA to dissolve.
- Once reconstituted in TE buffer, the DNA should be stored at -30 degreeC.
Related Information
Primers for PCR of rRNA gene
| Bacteria 16S rRNA | |
| Name of primer | Sequence |
|---|---|
| EB-20F | 5′ AGTTTGATCCTGGCTC 3′ |
| EB-350F | 5′ TACGGGAGGCAGCAG 3′ |
| EB-1100R | 5′ AGGGTTGCGCTCGTTG 3′ |
| EB-1400R | 5′ ACGGGCGGTGTGTAC 3′ |
| EB-1530R | 5′ AAGGAGGTGATCCAGCC 3′ |
Reference of primer
|
|
| Archaea 16S rRNA | |
| Name of primer | Sequence |
| A-20F | 5′ TCCGGTTGATCCTGCCG 3′ |
| A-340F | 5′ CCCAGGCCCTACGGG 3′ |
| A-520R | 5′ GTATTACCGCGGCGGCTG 3′ |
| A-1100R | 5′ CGGGTCTCGCTCGTT 3′ |
| A-1400R | 5′ GACGGGCGGTGTGTGC 3′ |
Reference of primer
|
|
| Fungi 26S rRNA | |
| Name of primer | Sequence |
| ITS5 | 5′ GGAAGTAAAAGTCGTAACAAGG 3′ |
| ITS4 | 5′ TCCTCCGCTTATTGATATGC 3′ |
| NL1 | 5′ GCATATCAATAAGCGGAGGAAAAG 3′ |
| NL4 | 5′ GGTCCGTGTTTCAAGACGG 3′ |
Reference of primer
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