University of Pennsylvania
DNA Sequencing Facility
Template (Plasmid) Preparation Including Transformation & Purification
Template DNA can be prepared and/or purified by the facility staff. Plasmid, cosmid, lambda phage, and BAC preps are offered through the Sequencing Facility. PCR reactions and purification using commercial columns is also offered by the facility. Plasmid minipreps are done on the Qiagen Qiacube. These preps yield 5-10 μg DNA for high copy number plasmids. If used for sequencing, the Facility guarantees the results, i.e. we will repurify and repeat sequencing at no additional charge until successful sequence is obtained.
Transformation service is provided is to make a large prep from a small amount of left-over DNA.
The facility also offers reliable treatment of marginal or bad templates to make them suitable for automated sequencing. This involves RNase treatment followed by column purification or phenol-chloroform extraction. This treatment improves quality of template, producing more reliable sequence and longer reads. For primer walking projects we recommend that a substantial amount of DNA (nominally 4-5 μg per kb for each direction) be treated this way and be used for the whole project.
Templates prepared or purified by the facility staff are guaranteed to produce good sequence with standard, reliable primers, barring sequence specific problems (GC rich templates, secondary structure, internal repeats). The facility also provides free training in template preparation and troubleshooting, at users' request.
Please also consult the template preparation guidelines recommended by the Facility.
The DNA Sequencing Facility is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.