"
Contents |
Preparation of the DNA sample for LovTAP sequencing
Protocol: DNA Sequencing Sample Preparation
Microsynth
[http://microsynth.ch Microsynth] is a Swiss sequencing company that has a pick-up service at EPFL, which means the sample doesn't need to be sent anywhere. It also has a [http://microsynth.ch/de/10190/Hints-and-Tips.html#stpl library of standard primers], in case there is some kind of common sequence in the vicinity of the sequence of interest that could be used as the starting point for Sanger sequencing.
Requirements
- Design the sequencing primers that should start approximately 50 bp upstream and downstream of your sequence of interest. If there is a standard sequence in that area, pick a pre-made primer from the [http://microsynth.ch/de/10190/Hints-and-Tips.html#stpl standard list]. You can also order the primers from some other company and send them along with the DNA sample.
- Test your primers by running a virtual PCR with [http://serialbasics.free.fr/Serial_Cloner.html Serial Cloner] or any other software on your sequence. If you get the expected product, they are correct. Log in to the [http://microsynth.ch Microsynth website], place your order and print out the primer barcode.
- Prepare the DNA of interest according to [http://microsynth.ch/de/10179/Economy-Run.html Microsynth guidelines]. The plasmid concentration should be 80 ng/µl, and the total volume shall be 15 µl. Therefore, you need to take 1.2 µg of DNA from your original sample, and complete it to 15 µl with either pure water, either 10 mM Tris-HCl (pH 8), either 10 mM Tris-HCl (pH 8) with a maximum of 0.01 mM EDTA, depending on your needs. Use the tubes that are recommended by the company (screwcaps). Make two identical 15 µl samples for every DNA sample you want to sequence, one will be used for the forward primer, and one for the reverse one. Put prepaid Microsynth stickers on them.
- Leave the DNA sample and the primer barcode in the EPFL pick-up area (SV building, level 0). They will be shipped off to Microsynth.
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 water. 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]
