Protocol: Big Dye Terminator Mix* 16ul ThermoFidelase** 1ul Primer (18-24mer) 15-30pmoles Genomic DNA Template 3-6ug Final Reaction Volume 40ul Cycle Conditions: 95 degC - 5 min. initial hold 45 cycles of 95 degC - 30 sec. 55 degC - 20 sec. 60 degC - 4 min. 4 degC hold Purify through G-50 spin columns and load under standard gel conditions *PE-ABI #4303150 for the 1000 reaction kit Description: TF,KIT BTD RR-1000 **Fidelty Systems Inc. (301) 527-8250 http://www.fidelitysystems.com
|Reaction "size"||Big Dye Mix (ul)||20-30mer
|Purifed Bacterial Genomic DNA Template(ngrams)||ThermoFidelase (ul)||H20 to final volume of||Number of Cycles|
|C. Heiner's 2x reaction||16 ul||15-30||3000-6000||1 ul||40 ul||45|
|Roe-Lab 1x reaction||8 ul||15-30||3000-6000||1 ul||20 ul||99|
Modified Bacterial Thermo-Cycle Conditions :
Note #1: Increasing the number of cycles from 45 to 60 to 80 and eventually to 100, almost linearly improved both the signal and read length. Increasing the annealing temperature from 50 to 55degC and the extension temperature from 60 to 65degC also improved the signal and read length but to a lesser extent than increasing the number of cycles. Because of the increased number of cycles, these incubations only should be done overnight.
Note #2: ThermoFidelase improves the signal and read length with bacterial genomic templates for all primers tested.
Note #3: Although some primers work better than others and we have no clue why, it is clear that any proposed primer should be screened against the already known sequence data for the entire know portions of the bacterial genome, as well as sequencing vector sequences. Thus, to prevent the primer from binding to multiple places on the target clone, the primer sequence should be compared to all the contigs in the target clone database. Only those primers with a homology of less than 12/20 Smith Waterman Identity elsewhere on the target clone should be used for custom primer directed sequencing. The PrimOU computer program is available from our informatics group that has been modified from the SW Medical Center's Primo program, to allow screening against multiple contigs in a phred/phrap database at various stringencies. In addition, typically we choose primers with either a G or C on their 3'end and a roughly even distribution of each of the 4 bases or a slightly higher G/C content (i.e. 1-2 more G's or C's than A's or T's to shift the Tm slightly higher)
Note #4: As one increases the primer length, the annealing temp. can be increased so too can the extension temperature. We have tested 20,25,30,3 5 mers on bacterial genomic templates as well as increasing the extn temp from 60 to 65 to 72degC and the annealing temperature from 50 to 55, 60, 65, 70degC.
Our conclusion is that:
Heiner CR, Hunkapiller KL, Chen SM, Glass JI, Chen EY
Sequencing multimegabase-template DNA with BigDye terminator chemistry
Genome Res 1998 May;8(5):557-61
Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of >700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial genome sequencing projects without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.
Bruce Roe, firstname.lastname@example.org