The HotShot is the ace in SEQLABs history. Although fluorescent colour band sequencing has been available since the 90s and was offered by various sequencing centres the costs remained so high that many smaller laboratories and university departments preferred to use their own resources. This involved radioactive methods as a rule, and the yield, following two days of heavy work, was information on a modest 250 bases.
The breakthrough occurred in 1999 with SEQLABs HotShot – primer and the DNA for sequencing were sent ready mixed – and the savings in work time were passed on to the client in the form of reduced costs. Thus sequencing within ones owns four walls became even less attractive economically. Since it was not possible to repeat the cycle sequencing a suitable CS protocol had to be developed to enable the sequencing of DNAs including those of simple secondary structures to be carried out successfully at the first try.
This protocol has been available since 2000 and constantly delivers good results. 97% of clean DNAs with no excessive secondary structures deliver 99% exact readings.
What we need:
For HotShot or extended HotShot sequencing reactions of plasmids please pipet 600-700 ng plasmid DNA and 20 pmol primer into a 0.2 ml flat-lid vial and fill it up to 7 µl. It is advisable to have 5-10 mM Tris pH 8.0 in the sample.
For HotShot or extended HotShot sequencing reactions of PCR-products please send us: The amount of DNA (in nanograms) is calculated by dividing the PCR-product-length by 4. Put this amount into a 0.2 ml flat-lid vial and add 20 pmol primer. Total volume should be 7µl and it is advisable to have 5-10 mM Tris pH 8.0 in the sample.
Please number the HotShot samples consecutively (e.g. 1 to 5 or 10 to 15). The reaction tubes should be labeled on the lid with a permanent felt marker. Please do not use removable labels or 8'-stripes.
Please protect your samples by using an extra plastic tube and a stable envelope.
Please note also our PDF-brochure: "INFOS and TIPS"