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Preparation of the DNA sample for LovTAP sequencing
Protocol: None
Forgot to insert protocol.
Goal
As no LovTAP expression was seen on the Western Blot, we decided to start at the basics and to check if the Maxiprep we had still matched the original LovTAP sequence. The sample had to be sent to Microsynth AG for Sanger sequencing (incorporation of fluorescent nucleotides).
LovTAP sequencing primers
The forward primer is the T7 primer from the Microsynth standard primer list, 40 bp upstream of LovTAP. The reverse primer is a custom one, 50 bp downstream of LovTAP. They were designed like normal PCR primers and verified with [http://serialbasics.free.fr/Serial_Cloner.html Serial Cloner] by running a virtual PCR, which gave a 985 bp product.
LovTAP preparation
The concentration of our LovTAP Maxiprep is known to be 294.5 ng/µl. We needed 1.2 µg (1200 ng), according to the [http://microsynth.ch/de/10179/Economy-Run.html Microsynth guidelines]. So we took 4 µl of our sample and completed the volume to 15 µl with ____________. The sample was then shipped to Microsynth, along with the primer information.
Links
[http://microsynth.ch/de/10179/Economy-Run.html Microsynth Economy Run description and requirements]
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