Ulrike Heberlein

DOUBLE STRANDED SEQUENCING

Minilysate DNA:
double-stranded sequencing;
1) Prepare DNA using either alkaline lysis or STET/boiling protocols from 1.5 ml of saturated bacterial culture. (I have found that DNA isolated from strains HB101 or TG-2 sequences well, whereas DNA isolated from TG-1 doesn't work as well). Never freeze the bacterial pellet before isolating the DNA!!! Before denaturing, clean the DNA by consecutive phenol:chloroform and chloroform extractions. Precipitate with 2.5 volumes of ethanol in the presence of 0.3M NaOAc and wash DNA pellet with 70% ethanol. Resuspend in 25µl of TE.

2) To 5µl of the cleaned DNA add 0.5µl of 0.5mg/ml RNAse A and incubate at room temperature for 2-5 minutes.

3) Add 5µl of 1M NaOH (make up from 10M stock about once a month) and incubate at room temperature for 5 minutes.

4) Neutralize by adding 3µl of 3M NaOAc pH 5.0.

5) Add 7µl of a ~1pmole/µl solution of primer (MW is~330g/mole/base; absorbance of a 1mg/ml single stranded DNA solution is ~25).

6) Precipitate with 75µl of 100% ethanol at -70°C for 15-30minutes (or in dry ice for 5-10 minutes). Spin in a cold microfuge for 20 minutes. Wash the pellet with 70% ethanol and dry (the pellet should be visible).

7) Resuspend thoroughly in 8µl of water, add 2µl of Sequenase; buffer and anneal as usual. Follow Sequenase instructions.

CsCl purified DNA:
€ Use 2-3µg of DNA in a 5µl volume. Denature with 5µl of 1M NaOH for 5 minutes at room temperature and follow protocol described for minilysate DNA.