Single Stranded DNA Sequencing

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Mon Sep 13 22:10:17 EST 1993


I ask because I've used protocols for ds sequencing with go 
>sucess until recently. Lately, I had alot trouble with my ds templates. I 
>remember reading that Sequenase 2.0 prefers ss templates. Is it worth it to 
>switch? Antone have good protocols? Thanks in advance.
>
>Picard

cooperj at nhlbi.nih.gov (John Cooper) asks:

>Does any one know if single stranded sequencing is really superior to ds 
>sequencing? 

I think that you get the best consistency with single-strand templates.  DS
templates exhibit more variability.  It's probably to have highly purified
DNA.  Although "CsCl-prepped" DNA is obviously the best, for  preparing
multiple templates, we DNA prepared with the normal alkaline-lysis miniprep
procedure and PEG precipitate (as described, for example, in the Molecular
Cloning manual). This purified DNA is then denatured according to the
latest guidelines in USB's Sequenase manuals which suggest alkaline
denaturation _at 37oC_. 

>Antone have good protocols?

I've used the following alternative, highly simplified protocol (from
friends while at Princeton) for the preparation of ss DNA templates from
recombinant phagemid vectors (e.g. pBlueScript):

1.  Inoculate 1.5 ml 2x TY (or other rich broth) with a toothpick-full of a
colony and 2 ul of helper phage (e.g. f1 R408 @ 10^11 pfu/ml).  Grow
overnight at 37oC with _vigorous_ agitation.  

2.  Next day, pour broth into 1.5 ml centrifuge tube.  Spin 3 min. at >13,000xg.

3.  Transfer supernatant to new 1.5 ml tube with 260 ul 2.5M NaCl, 20% PEG
and 8 ul 5 mg/ml RNAse.  Incubate 15 - 30 min. at room temp.

4.  Spin 5 min. at >13,000xg.

5.  Decant supernatant.

6.  Spin again quickly to collect remaining liquid.  Remove residual PEG
solution with pipetter.

7.  Resuspend pellet in 100 ul T.E.

8.  Extract 1x with 50 ul phenol (DO NOT re-extract).

9.  Precipitate with 1/10 vol. 3M NaAcetate, pH 5.2 and 2.2 vol 100% EtOH;
-20oC for >1 hour.

10. Wash and dry pellet (may be hard to see).

11. Resuspend in 20 ul T.E.  Store at -20oC.

12. Use 5 ul for each set of sequencing reactions.  Use 2 ul to check ss
template DNA on agarose gel.

Let me know if you have any questions with this protocol.  

Good luck,
Bill Morgan
William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu




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