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Contents |
Meeting with professors and T.A.s, PCR debugging, pMP-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.
We discussed the results obtained with the digestions performed during the weekend, had a meeting with professors and TAs and decided to sequence one of the minipreps of ligated LovTAP (pMP-LovTAP) which worked best. Samples where taken and sent to Microsynth with new primers.
After that, we found out that the plasmid we were trying to sequence was actually a religated pMP, and that our PCRs needed troubleshooting.
Other: Primer design for inserting LovTAP into pcDNA3.1 for Biobricking (turned out to be more complicated than expected), primer design for plasmid control, concept art, etc.
- Comments
The sequencing did not work with the reverse primer (BGH-rev).
Transformation for pMP-LovTAP cultures
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
E.Coli were transformed with pMP-LovTAP miniprep (L2) in the evening.
- Comments
It's not that it didn't grow... It grew WAY too much.
