Sequencing at MTU (Taught by Surendar Dhadi) PCR setup: x uL water 4 uL sequencing buffer 0.5 uL sequencing primer 100 ng plasmid (if total is 4 kB) or 200 ng plasmid (if total is 8 kb) 0.5 uLBig Dye -------20 uL Note: Plasmid must be very pure: 260/230 must be above 1.5 260/280 must be above 1.8 o o 2 min 95 C, 99 cycles of (26 sec 95 C, 35 sec 46, 3 min 60 oC), 10oC Store at -20oC for 6 months if desired Sequencing reaction purification: - Reuse specific sequencing columns stored in 1M HCl at least o/n to destroy DNA, spin out HCl, wash 2 x with 500 uL water, ready to load with Sephadex G-50 beads 5M HCl: 15 mL concentrated + 85 mL water 1M HCL: Dilute 5 M 1:5 - While stirring Sephadex suspension with a magnet, add Sephadex 50 suspension to the columns, let stand a few seconds to let beads sink, remove water from top (put it back into Sephadex bottle, add more suspension until the columns are almost full with beads - Spin the columns 3 min at 0.9 g - Add one more time Sephadex 50 suspension - Spin 3 times 3 min at 0.9 g to remove all the water (Orient the columns in the same way into the centrifuge to not destroy the slant of the Sephadex structure) - Load the PCR sequencing reaction in the middle of the Sephadex surface (usually 18 uL are left) - Place the column into a fresh 1.5 mL tube - Spin 3 min at 0.9 g (orientation of the column should be followed) - Now about 13 uL very pure PCR reaction are left - Add 7 uL water to regain a volume of 20 uL Loading the Sequencer: - Use 0.5 mL tubes, put 20 uL clean sequencing reaction in them, and cut the lids off - Up to 8 reactions can be ran without rubber lids - If more reactions shall be run, rubber lids have to be put onto the tubes to prevent evaporation - Open both doors of the machine - Check if all buffers and other liquids are filled enough to sequence your amount of samples, check with someone in Rama’s lab) - A capillary lasts for 300 runs - Use only Pop4 polymer, never Pop6 - 500 uL Pop4 are loaded in glass syringe, gives 50 – 60 sequences - Strictly avoid air bubbles in the capillary - 2 buffer reservoirs exist, buffer load good for 30 – 40 sequences or 2 – 3 days, then change buffer - Push TRAY button -> Tray moves - Add sequence reactions in a row to the tray, starting A1, the A3, then A5, … - Push TRAY button again, reactions go inside - Close both doors Program the sequencer using the computer: - Open “310 Data Collection” software - File>New>Create New, choose “Regular Sequencing” “Sequence Sample Sheet 48 Tubes” - Enter sample name (Start with A1, then A3, then A5, …) - Choose “Matrix file”: <none> - File>Save: Give file name with date - File>New, choose Sequence Injection List (double-click), Sample Sheet (retrieve the file you just saved from a huge list) - Pull down proper module: P4StDSeq(1ml)E.md4 - Fill down module for all sequences: Mark all (click on the header), then Ctrl-D - Run time (min) leave at 32 if ,500 bp or set to 40-45 if 1 kb or more - Injection seconds: Go up to 80 (more sample will loaded, it’s good because we use so little BigDye) - Window>Status - Click the Run button, note the date and time (you will need it to find your sequences later - It takes 1 h per sample Retrieve the data on the computer: - Start “Sequencing Analysis 5.2” software - User name = geneseq - Password = geneseq - File>Add sample: Choose your folder according to the date and time when your samples were ran - Press OK, and my samples will be displayed - (Select all, click “Clear” button to make the window clear) - Select: Dye Set Primer: KB_310_POP4_BDTV3_36std.mob (from pull down menu) - Select: Matrix File: 310Matrix5.mtx (from pull down menu) - Make selections for all samples (use the Ctrl-D trick as mentioned above) - Check all boxes in front, and then hit the green Run button - Colors indicate quality: Blue = excellent, green = good, yellow = bad - Go to Sequence box, mark all, copy, paste in WordPad (There must be a way to export the whole data set as ab1 files or something like that)