User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/16

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Bench work Glycerol stocks 750μL pTXB1His/DH10B QC colony #2 culture from yesterday + 250μL 60% sterile glycerol → tube #70 750μL pTXB1His/DH10B QC colony #3 culture from yesterday + 250μL 60% sterile glycerol → tube #71 750μL pTXB1/DH10B colony #1 culture from yesterday + 250μL 60% sterile glycerol → tube #72 → -80°C Miniprep O/N cultures from yesterday Cultures grew about 17h Followed standard miniprep protocol Quantify [minipreps] Done with Daniel Catt 95μL water + 5μL DNA from miniprep → read A260 and A280 NheI digest performing sequential double digest, so use optimal buffer for SapI instead of NEB buffer 2 modify NheI digest protocol as follows Tube sterile H2O 10X NEB4 100X BSA DNA NheI (10 kU/mL) pTXB1His#1 34.5 μL 5 μL 0.5 μL 5 μL of 102 μg/mL 5μL pTXB1 34.5 μL 5 μL 0.5 μL 5 μL of 82 μg/mL 5μL BSA PCR 24.5 μL 5 μL 0.5 μL 15 μL directly from PCR reaction 5μL → 2h @ 37°C → 20' @ 65°C (it took awhile to get the block to the right temp, so they were on ice for a bit, then at ~70°C before the block reached 65°C) Note: I should have done a PCR cleanup on the BSA PCR sample! I will repeat this. *Kathryn Muratore 10:20, 20 June 2011 (EDT): In retrospect, I'm not sure that I even used the correct tube here *Kathryn Muratore 16:26, 20 June 2011 (EDT): I did use the right tube, as seen on the gels later that week *Kathryn Muratore 10:33, 28 June 2011 (EDT): → store @ -20°C Results {{#widget:Google Spreadsheet		key=0Aph9WCMwmkfMdF9PNG53aGllblpSLW1zdmpqcVR0TlE }}