Method prepared by
Grace Panganiban: firstname.lastname@example.org
ABI Sequencing Dos and Don'ts
1) Template preparation
You need 0.1-1.0 ug of plasmid DNA in < 9.5 ul, and it must be very clean.
Both host strain and DNA isolation methods affect template prep.
DNA from DH5-alpha prepared using the Qiagen plasmid minipreps (with P1, P2, N3, PB, and PE buffer chemistry) worked well for me.
2) Primer design.
The ideal primers have a Tm of ~60oC, have a GC content of 50-60%, are 20-25 bases long, and have no runs of any bases. However, I still got readable sequence using a 22 base primer with only a 35% GC content.
3) Reaction components.
We are switching from a sequencing mix which uses the CS-version of Taq polymerase to one which uses the FS-version of Taq polymerase. The FS enzyme gives more uniform peak heights, which makes it easier for the base-calling software (and for you) to read the sequence. The FS enzyme also requires less template, and can tolerate greatly reduced amounts of nucleotides in the mix. In some cases it is then possible to load the reactions directly (i.e. without a Centrisep column). It is definitely possible to replace the Centrisep step with an ethanol precipitation, although the first 50 bases may be weaker. Freezing and thawing the FS/nucleotide mix is very hard on it. The mix must be aliquoted when it arrives (liquid and on wet ice) and before it is frozen. According to George Mayhew (Blattner lab sequencing manager), it is better to store the mix at 4oC for up to 1 month, rather than to refreeze it.
4) Cycling parameters.
The parameters in the ABI handbook are okay, but people in Horticulture and McArdle have found it better to use the parameters described for the model 480 thermocycler, when using the model 9600 thermocycler. Method #40 on our 9600 is set for the 480 conditions.
5) Cleaning up the reactions
As mentioned above, you can use either an ethanol precipitation or a Centrisep column to get rid of unincorporated nucleotides. You need to give the sequencing facility a dried down reaction. If you use a Centrisep, you can Speedvac (no heat) your sample dry. If you use an ethanol precipitation, be sure to do a 70% wash before air drying or speedvacing the pellet.
6) Sequencing facility procedures
You must fill out a request form by noon on the day before your desired run, and you must fill out an invoice by noon on the day of your run (it's fine to do these at the same time). The forms can be found outside the sequencing facility in the basement of Genetics, and should be dropped of in appropriate in boxes at the same location. Gels are run in the afternoon/evening, so samples (clearly labelled and in plastic baggies) should be left at the sequencing facility no later than 2 pm on the day of the run.
7) Retrieving your data
George puts an image of the gel, the histogram files, and text files of the basecaller derived sequences on a Genetics server. These are usually available by noon of the day following the run. Once you have accessed the Genetics Appletalk zone using Appleshare, click on the server named "Moldoveanul", and enter "carroll" as the user, and password available from lab (all lower case) as the password. You then have access to the "Carroll" folder, and should transfer your files to one of our computers asap.