Cycle Sequencing Protocols—BigDye & DYEnamic ET
This page contains current as well as no longer used protocols (numbering helps us keep track of modifications, changes, etc.)
Protocol 1.5: Recommended BigDye starting point.
DNA (ds = 100-200 ng for < 20 kb plasmid, PCR = 10ng/100 bp, lg. plasmids, BACs, cosmids 500 ng-1 ug)
Primer (5-20 pmol; for PCR fragments stick with 5)
Enzyme Reaction Mix: 2 ul BigDye plus 3 ul 5X buffer, H2O to 20 ul. Big Dye itself is suspended in 2.5X buffer. (note change from previous protocols)
Begin at 95° 3' for "hot start", then 50 cycles of 96° 10", 58° 4’, finish with 72° 7'. The number of cycles can be decreased with minimal signal loss. In fact, we have also used the following protocol with success:
1.5a: 25 cycles of 96° 10” 58° 2’ . No hot start, no 72° finish. Good protocol if you’re in a hurry.
Protocol 1.6: DYEnamic ET official conditions
0.1-0.2 pmol template DNA, 5 pmol primer, 8 ul sequencing reagent, H2O to 20 ul.
25 cycles of 95° 20” 50° 15” 60° 1’ . DO NOT use an extended initial denaturation.
DYEnamic sequencing reagent can also be diluted using the Amersham Biosciences buffer, available for purchase here. In pilot experiments we have seen good results using 4 ul enzyme + 4 ul buffer or 2 ul enzyme + 6 ul buffer.
Reactions must be cleaned following sequencing to remove unincorporated fluorescent nucleotides. We can do this for a charge (select one of the cleanup options from the sign in menu). To clean the sequencing reaction following cycling, we use (and recommend) magnetic beads from Agencourt (www.agencourt.com). For what we do, see the document entitled “Facility procedures” at https://dna.biotech.wisc.edu. You can also use spin columns or alcohol precipitation (see the document entitled “Non-bead cleanups” for protocols at https://dna.biotech.wisc.edu). DYEnamic ETs can also be cleaned similarly, but talk to us first regarding protocol modification.
BACS and related larger templates require more template (1-2 ug) and primer 25-50 pmol. We have BAC specific protocols available in the facility. If secondary structure or high GC is a problem, the extension step temperature can be raised up to 65°, and DMSO can be added to a final concentration of 5%. Very stubborn GC regions can also be sequenced with a specially formulated BigDye mix containing GTP and/or other additives. We can give you enough of this reagent to do a couple of reactions for free. Other problem templates include polyT or polyA regions, and other dinucleotide repeats. There are strategies to get through these, talk to us for info.
Note that these are SAMPLE protocols, that work in most (but not all) applications. It may be necessary to manipulate several variables to get acceptable results. Template quality is the number one factor in getting good data, but you may need to also vary annealing or extension times, denaturation temperatures, primer composition, etc. to get what you want. We have control template and primers available (see the “Available Controls” document at https://dna.biotech.wisc.edu for a description of the controls available and what they tell you), and can talk to you about troubleshooting if difficulties persist. Call us if you have any questions about your data—there is information in the electropherogram that can help troubleshoot. Sometimes a simple re-run can lead to better results.
Sequencing Primer Design:
Consult the document entitled “Primer Considerations” at https://dna.biotech.wisc.edu for several URLs featuring free primer design utilities, and for a description of our rule of thumb guidelines for sequencing primer design.
Help lines: Local: 263-9882 or 9880, or jrhyman@wisc.edu; ABI: 800-831-6844, then go through the menu.
THE PROTOCOLS BELOW ARE FOR OLDER VERSIONS OF BigDye AND BUFFER
Protocol 1.0 :
Set up reaction in 200 ul PCR tubes:
DNA (ds = 100-400 ng, ss = 100-200 ng, PCR = 10ng/100 bp)
Primer (5-20 pmol; for PCR fragments stick with 5)
Enzyme Reaction Mix: 4 ul BigDye plus 4 ul 2.5X buffer (5X is 400 mM Tris pH 9, 10 mM MgCl2 H2O to 20 ul). Amount of BigDye can be lowered to 2-3 ul, depending on your signal strength. Add buffer so total volume = 8 ul
Begin at 95° 3' for "hot start", then 35 cycles of 95° 20", 45° 30", 60° 4', finish with 72° 7'.
Protocol 1.1: Same reaction set up as above, but cycle as follows:
Begin at 95° 3' for "hot start", then 50 cycles of 95° 20", 45° 30", 52° 4’, finish with 72° 7'. (Good procedure for AT rich templates)
Protocol 1.2: 250 ng ds plasmid, 10 pmol primer, 2 ul BigDye mix, H20 to 7 ul
Cycle at 95° 30", 50° 20", 60° 4' 25 to 99 times. (no initial denaturation or final extension)
We haven’t tried this one, but it is from Bruce Roe’s group (via Peter Evans) and has given good results.
Protocol 1.3: DNA (ds = 200-500 ng, ss = 50-100 ng, PCR = 30-90 ng), primer 3. 2 pmol
Cycle at 96° 10" 50° 5" 60° 4' 25 times. No initial denaturation or final extension. These are the "official" ABI conditions.
Protocol 1.4: Same reaction set up as 1.0, but cycle as follows:
Begin at 95° 3' for "hot start", then 50 cycles of 96° 10", 54° 4’, finish with 72° 7'. Good quicker protocol.