7/15/2023 0 Comments Microsynth single tube sequencingLook for the best pair of restriction sites, ideally with similar digestion temperatures and times. The bands for NFAT are slightly weird, but for the rest, BioBricking PCRs finally seem to work! PCR to amplify as much inserts as possible for Biobricking, and to determine once and for all what the correct BioBricking annealing temperatures are.Įight 20μl reactions per Biobrick insert were made, so in total 32 main reactions and 1 control reaction (pMA-LovTAP with LovTAP_fwd and LovTAP_rev). Don't forget to sequence your final product (this could be your final plasmid): you really don't want to lose a few weeks because of a "corrupt" plasmid. Avoid primers with strong secondary structures.If you're not sure what the correct Tm is, consider using a gradient PCR. Primer Tm calculation is a less exact science than it should be (just test several tools and compare their results).Primers should have similar Tms (less than 5☌).Don't forget positive and negative controls.If the reactions have different primers and/or template, add the polymerase right after the dNTPs, split the mastermix and add the rest.Avoid taking the Phusion-HF polymerase out of the freezer (only take it out briefly when you need to add it).Thaw the HF-Buffer, DMSO and dNTPs before making the mastermix.Once you've finished, you should run the resulting products on a gel Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips). Master mix, the composition for one tube is:ġX Mastermix 20μl reaction, add in this order PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). When you've received the primers, prepare them and make sure you've got your Primer design can be done by hand, or byĭone, order the primers (in our case, we ordered from them IDT). (and the design of the relevant primers). The first step is the selection of that region PCR is a reaction that makes it possible (and relatively easy) to amplifyĪ certain region of DNA. The sequencing sample of C6 was shipped off to Microsynth. Leave the DNA sample and the primer barcode in the EPFL pick-up area (SV building, level 0).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. Use the tubes that are recommended by the company (screwcaps). 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. The plasmid concentration should be 80 ng/µl, and the total volume shall be 15 µl. Prepare the DNA of interest according to Microsynth guidelines.Log in to the Microsynth website, place your order and print out the primer barcode. If you get the expected product, they are correct. Test your primers by running a virtual PCR with Serial Cloner or any other software on your sequence.You can also order the primers from some other company and send them along with the DNA sample. If there is a standard sequence in that area, pick a pre-made primer from the standard list. Design the sequencing primers that should start approximately 50 bp upstream and downstream of your sequence of interest.It also has a 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. 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.
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