TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.
In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.
Konecki
D S
DS
Center for Medical Genetics, Marshfield Medical Research Foundation, WI 54449, USA.
Phillips
J J
JJ
D004274:DNA, Recombinant / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D010957:Plasmids / Q000191:economics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA / Q000379:methods; D013698:Templates, Genetic
D004274:DNA, Recombinant