ABI SEQUENCING PROTOCOL

version no. 1

(prepared by Richard Clark, 7/28/97)

Preparation of template:

ABI sequencing requires high quality DNA template. Several miniprep kits are available for preping ABI quality DNA. In our hands the UltraClean Mini Plasmid Prep Kit (cat. no. 12300, Mo Bio Labs, 800-606-6246) has worked very well, and is the least expensive ABI quality kit with which we are familiar ($0.88/prep). It takes about 45 minutes to prep 18 templates, and is easier to use than some other preps.

1. Grow up plasmid in 2.5 mL cultures (with pBSSK we use 75 ug/mL amp).

2. Prep DNA exactly according to the manufacture's instructions using 1.8-1.9 mL of cultures.

Note: Be sure to elute DNA in 50 uL ddH2O (i.e., still water) and not in TE!!! Store at -20oC.

Setting up and Running Reactions:

We use a DNA sequencing kit (cat. no. 402079) from Perkin Elmer (order from the Foster City ABD unit).

The enzyme mix (FS mix) will come on ice--aliquot immediately upon arrival and free at -20oC. Repeated thaws will cause significant loss of polymerase activity, and it is best to let the mix only thaw once (although this gives the best results, thawing 2-3 times still gives okay sequence). An 80 uL aliquot is enough for a 36 lane gel. The mix is light sensitive, so throughout the sequencing procedure keep in the dark as much as possible.

Set up rxns as follows in thin walled PCR tubes:

1.5 uL DNA preped with UltraClean kit (or 0.125 ug

or CsCl preped DNA)

0.5 uL primer at 1.6 uM in water

1.0 uL ddH2O (still water)

2.0 uL FS enzyme mix (thawed in the dark at room temperature)

Add the FS mix last and pipet up and down several times to mix. Cover with 40 uL mineral oil and put in PCR machine (do not hot start). Typical cycle program is:

96oC 10 sec |

50oC 5 sec | 25X

60oC 4 min |

4oC hold

Notes: It is easiest to add DNA to a bunch of tubes and next add the other components (including FS enzyme mix) from a premix. How much DNA to put into the reaction will vary as a function of how well the plasmids grow. For preps of inserts in pBS 1.5-1.75 uL of DNA per reaction works well (although specing DNA would be best, this is a lot of work for a large scale sequencing project). If using CsCl preped DNA a minimum of three precipitations is required after the normal CsCl protocol to chase away residual salts, followed by resuspension in ddH2O.

Precipitating rxns:

Add 15 uL of ddH2O to the 5 uL of rxn in PCR tube and mix by pipeting up and down several times. Next remove all 20 uL to a 1.5 mL eppi tube, and add 50 uL of 95% (or 100%) ethanol and 2 uL of 3 M sodium acetate pH=5.2 and mix well. For a large number of samples it is best to premix the ethanol and sodium acetate. Put on ice for 12.5 minutes, and then spin for 26-30 minutes in a microfuge at 14K. Remove ethanol and add 150 uL of 70% ethanol and let sit for 1-2 minutes. Remove as much of the 70% ethanol as possible with a P200. Speed vac for 8 minutes without heat.

Note: After speed vac, there will generally be 0.5-1.5 uL of mineral oil in the bottom of the tube. The mineral oil will not hurt anything.

Loading samples:

Make formamide/blue dextran/EDTA loading buffer (100 uL formamide, 20 uL blue dextran-EDTA). Blue dextran/EDTA solution is from Perkin Elmer (cat. no. 402055) Add 1.6 uL of loading buffer per tube, resuspend by vortexing several times. 2 minutes at 80oC and load 1.5 uL onto ABI gel, being very careful not to get any mineral oil in the pipet tip. The mineral oil will clog the pipet tip.