Cleave 5' Overhanging Ends:
          5 µl (1 µg) ds-cDNA [use only 1/2 of 2µg; store rest at -20°C]
          2 µl 10X T4 polymerase buffer (per Maniatis)
          12 µl H₂O
          4 µl dNTPs (1µl each of 2 mM stock)
          0.5 µl T4 DNA polymerase (6U/µl stock; 3U)

Incubate 5 min, 37°C. Add 1 µl of 0.5M EDTA. Extract once with phenol:CHCl3. Precipitate once with ammonium acetate/ethanol, wash with 80% ethanol, and dry the pellet.


Methylate to Protect EcoRI Sites in cDNA:
This is an optional step if you are adding EcoR I linkers to a cDNA that may contain internal EcoR I site(s). Omit this step if there is no EcoR I site in the insert you are studying. The basis for this protocol was published by Greene, et al (J. Mol. Biol. 99:237-261, 1975), and by T. Maniatis et. al, (Cell 15:687, 1978).

     1-2 µg ds-DNA
     100 mM Tris-HCl, pH 8.0
     10 mM EDTA
     5 µM S-adenosyl-methionine
     1 U EcoRI methylase

Incubate for 1 hr at 37°C, then phenol extract. (If you are methylating oligonucleotides, incubate at 12°C), Precipitate in ammonium acetate/ethanol. Wash with 80% ethanol, and dry the DNA. See Greene, et al (cited above) for assay to monitor methylation.


Ligate to Linkers:
1 µg ds-cDNA, dry
2 µl linker (1µg at 0.5µg/µl)
1 µl 10X ligation buffer
1 µl 100 mM DTT
1 µl 10 mM ATP
4 µl H₂O
1 µl T4 DNA ligase (1 U/µl stock)

Incubate at 15°C overnight. Add 1 µl of 0.5M EDTA and µl of TE. Extract once with phenol:CHCl3 and then precipitate once with ammonium acetate/ethanol. Dry the DNA.


EcoRI Digest:
1 µg ds-cDNA
22.5 µl 0.1X TE
2.5 µl 10X RI buffer
0.25 µl 100 mM DTT
2 µl EcoRI (20 U from a 10 U/µl stock)

Incubate 4 - 5 hrs, 37°C, then add 10 U EcoR I and incubate an additional hr at 37°C. Add 2 µl of 0.5M EDTA, extract once with phenol:CHCl3, and precipitate twice with ammonium acetate/ethanol. Wash the pellet with 80% ethanol. Dry the DNA. Resuspend in 6µl of 0.1X TE for ligation.

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