TempliPhi: A sequencing template preparation procedure that eliminates overnight cultures and DNA purification.
Preparing plasmid templates for DNA sequencing is the most time-consuming step in the sequencing process. Current template preparation methods rely on a labor-intensive, multistep procedure that takes up to 24 h and produces templates of varying quality and quantity. The TempliPhi DNA Sequencing Template Amplification Kit eliminates the requirement for extended bacterial growth prior to sequencing and saves laboratory personnel hands-on time by eliminating the centrifugation and transfer steps currently required by older preparatory methods. In addition, costly purification filters and columns are not necessary, as amplified product can be added directly to a sequencing reaction. Starting material can be any circular template from a colony, culture, glycerol stock, or plaque. Based on rolling circle amplification and employing bacteriophage Phi29 DNA polymerase, the method can produce 3-5 microg of template directly from a single bacterial colony in as little as 4 h. Implementation of these procedures in a laboratory or core sequencing facility can decrease cost on tips, plates, and other plasticware, while at the same time increase throughput.
Reagin
Michael J
MJ
Amersham Biosciences, 800 Centennial Ave., Piscataway, NJ 08855, USA. michael.reagin@amersham.com
Giesler
Theresa L
TL
Merla
Alia L
AL
Resetar-Gerke
Jeanine M
JM
Kapolka
Kinga M
KM
Mamone
J Anthony
JA
D004259:DNA-Directed DNA Polymerase; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D017422:Sequence Analysis, DNA / Q000379:methods*
D004259:DNA-Directed DNA Polymerase