dowload pdf Hello CTMers, please read the below and attached pdf how to upgrade our “plasmids” table and list of cell lines for us to work with GMO. Tables link. Please have a look at it and try to upgrade the table. I would like to review how is it going and update the instructions in case somebody hits a problem on thursday so that we have the table ready next tuesday. Thank you very much for your cooperation. ...

Logging in link to login website Volumes and concentrations link to instructions on the submission of plasmids Possible sample types are purified Plasmid DNA in following sizes & concentration: 2.5 - 25 kbp with 30ng/µl 25 - 125 kbp with 50ng/µl 125 - 300 kbp with 50ng/µl Please note: quantification measurement should be performed with a fluorometric method, like Qubit. If you only have a Nanodrop on site please double the amount. ...

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Stock Solutions FACS Buffer [5590uL per experiment] CD90-APC Antibody [10 uL] VIOLET L/D [0.5uL] IC Fixation Buffer [200 uL] Permeabilization Buffer 10x [1 mL] or 1x [4.198 mL] cTnT FITC [2 uL] PBS Distilled Water Preparation Detach your CMs and count them Divide your CMs into 2 eppendorfs (250-300k cells/eppendorf): 1st is UNSTAINED - 2nd is STAINED cells Centrifuge your cells 2.0 rpm 4min and remove the supernatant Add 500uL FACS BUFFER (EDTA 0.5 1:1000, FBS 0.5% in PBS) and centrifuge again Remove supernatant Add 100 uL FACS BUFFER to UNSTAINED eppendorf add 90uL to FACS BUFFER+10uL CD90-APC antibody to STAINED eppendorf Incubate on ice 30’ in the dark During incubation prepare L/D by adding 0.5uL of VIOLET L/D staining in 500uL of PBS (put on ice) At the end of CD90 incubation, add 600uL PBS to the eppendorfs containing your UNSTAINED and STAINEDED cells and centrifuge Remove supernatant Add 100uL PBS to UNSTAINED Add 100uL of L/D solution to STAINED Incubate on ice for 30’ in the dark At the end of incubation, add 600uL of PBS and centrifuge Remove supernatant and add 100uL of IC Fixation buffer to UNSTAINED and STAINED Incubate RT for 20 min in the dark Prepare Permeabilization Buffer 1X duiluting 1mL of Permeabilization Buffer 10X in 9mL of ultrapure distilled water At the end of incubation, add to your UNSTAINED and STAINED eppendorfs 600uL of Permeabilization Buffer 1X and centrifuge Remove supernatant Add 100uL of Permeabiization buffer 1X to UNSTAINED Add 98uL of Permeabilization Buffer 1X and 2uL of cTnT-FITC to STAINED Incubate 30’ RT dark Go to switch on the FACS and take FACS tube to transfer your samples Add to each FACS tube 2mL of PERMEABILIZATION BUFFER 1X At the end of incubation, transfer your cells in the relative FACS tube and centrifuge Remove supernatant and add 2mL/tube of FACS buffer Centrifuge, remove supernatant, and add 200uL of FACS buffer in each tube and go to read your results to FACS

Download a pdf of this protocol Use filter tips, double gloves, bleach waste, GMO desinfectant, desinfect all plastic material before disposal. Day 1 - HEK replate Re-seed HEK cells to obtain 10-20% confluence (ideally early in the morning) After HEK adhesion (late afternoon) proceed with transfection using either Fugene or Lipofectamine 3000 Day 1A - Transfection by Fugene Confirm plasmids concentration and prepare the mix of packaging plasmids plasmid #9: 1750ng plasmid #10: 3240ng plasmid of interest: 5000ng. Equlibrate Optimem to RT Vortex and spin down Fugene Dilute plasmid mix in 1mL Optimem, vortex, spin down Add 30uL of Fugene, mix by inverstion incubate 5-15minutes at RT Apply dropwise to 10cm dish Incubate overnight Day 1B - Transfection by Lipofectamine Confirm plasmids concentration and prepare the mix of packaging plasmids plasmid #9: 1750ng plasmid #10: 3240ng plasmid of interest: 5000ng. Equlibrate Optimem and Lipofectamine components to RT Vortex and spin down components Solution A: Dilute 35uL of Lipofectamine in 875uL of Optimem, vortex, spin Solution B: Dilute 20uL of P3000 from Lipofect kit in 875uL of Optimem, add your plasmids, mix by pipetting Mix A and B, incubate 10-15 min at RT Apply dropwise to 10cm dish Incubate overnight Day 2 - Exchange media Change media to 10 %KOSR in DMEM high Glucose (15mL) ...