BAC Sequencing Information

Quality of sequence derived from BAC (or P1 or other very large templates) depends on the quality of the template.  No news there!  The question is how to get the best template.  CsCl banding is still the gold standard for clean DNA—BACs prepared by this method will give great sequence.  However, this is not always practical.  Other methods that people across the country have used to generate BACs that sequence successfully include the following:

            Qiagen Large Construct preparation kit

            Qiagen Midi DNA prep kits (see modifications below)

            PsiClone mini preparation kits (not sure of manufacturer)

            Various in-house methods (alkaline lysis based)

This doesn’t mean no other methods or kits will work, the information is simply not available currently.

One user who was able to get good sequence from  BAC clones performed the following modifications of a Qiagen MidiPrep using Qiagen 100 tips:

            Use 100 ml of cells

            Resuspend in larger volume of P1, with the addition of 2 mg/ml lysozyme

            Incubate at room temp for 15’

            Add buffer P2, proceed normally from there until elution steps

            Elute with 5 1 ml aliquots of elution buffer preheated to 65°

            Precipitate large fractions containing the most DNA and resuspend in small

volume 10 mM Tris 8, typically 50-100 ul.

Concentration and purity can be ESTIMATED by spectrometry, but still should probably run a gel to see how things look.

Run Conditions:

            PE recommended conditions:

1-2 ug DNA, 10 pmol primer

95° 5’, then 35 cycles of 95° 30’’ 50° 20” 60 4’

However, as with normal sequencing a wide variety of conditions can work.  In an informal web-based survey, the following ranges of conditions were used by people to generate good data:

            Primer length:              18-24 nts

            DNA amount:              0.2-5.0 ug

            Primer amount:            10-30 pmol

            Reaction volume:         10-40 ul

            Denaturation:               95-98° for 10-60 sec

            Annealing:                   40-55° for 5-60 sec

            Extension:                   60° for 4-5.5 min

            Cycles:                        25-99

So clearly the template and primer quality are the determining factors, not the run conditions.  Finally, make sure your primer will work on the target sequence—several individuals have had problems with primers that never would have worked anyway.