Please be considerate of others and avoid overwriting the master files by following these directions carefully.Ĭreate a folder for your team (you and your partner) on the desktop of one of the 4 Mac computers in the lab. NOTE: IF you don’t follow the directions in the first two paragraphs below for creating your own folder and for copying the files in the master practice folder (rather than working with the master files), you will RUIN the course master files for all other students coming after you. Once you have obtained the most accurate sequence information possible for the mutagenized cI gene, you will compare them to wild type cI and hypothesize the source of a functional defect in the cI gene product (lambda repressor protein). You will scan for disagreements between the fragments and make decisions about the accuracy of the sequence by observing the peaks in the chromatogram of each of the sequenced fragments.
Sequencher software software#
The software program, Sequencher 4.5, will be used to fit the product of the sequencing reactions (the separated fragments of different sizes created in the thermal cycler) together and assemble the total gene sequence. These primers were both expected to anneal slightly upstream from the cI gene in a pBR322 based plasmid, and then sequence in a clockwise direction. The only difference is that one primer (30mer) had more bases than the other. GOAL: Assembling and editing sequenced parts of a plasmid & comparing it to the sequence of a wild type gene of interest.Īlthough it is preferable to confirm the sequence of a gene in both directions using forward and reverse primers for each template, these sequencing reactions used two forward primers: EcoRI clockwise 15mer and EcorRI clockwise 30-mer. PLEASE FOLLOW THE DIRECTIONS CAREFULLY, so that the students who come after you will find the files they need in a useable, unchanged form. DO NOT transfer or remove any files from the lab folder on the desktop! Instead, please copy and paste the files you want to use into a new folder that you will create, as described in the beginning of the directions. Use the instructions below to familiarize yourself with Sequencher software. The practice data can be found on the computers in the back of the lab.
Sequencher software how to#
You will be provided with sample sequencing data from previous experiments so that you can practice using this software and learn how to analyze your data. Practice using the DNA sequencing software Sequencher™
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