ࡱ> DFC[ bjbj 4*ΐΐ T#hKKKKKJJJ"""""""$$$'\"JJJJJ"KK"JfKK"J":!,!"K0"! ""0#!R''!"'!"dJJJJJJJ""JJJ#JJJJ'JJJJJJJJJ : Se-Met protein production protocol 1-16-08 by Brant from Kents notes from David W. Transform construct into B834 E. coli. LB o/n culture is fine. Do not want cells to grow too long (~10 hours) so start O/N late the night before growth. Make media day before so ready in morning. Use at least as much of each amino acid listed below: Ala0.86 gLys0.876 gVal0.85 gCys0.288 gLeu1.26 gTrp0.246 gAsp0.636 gAsn0.64 gPhe0.8 gGlu1.062 gPro0.552 gAdenine0.324 gGly0.72 gGln1.05 gGuanine0.36 gHis0.504 gArg3.3 gUracil0.27 gTyr0.44 gSer3.0 gCytosine0.264 gIle0.63 gThr0.57 gThymine0.3 g Dissolve in 1 L of H20 by stirring. Add a 50%/50% mixture of 8N KOH/ 10N NaOH until pH is ~12. Continue stirring. Dissolve in 1L H20: 8 g of NH4SO4 33.6 g of Na2HPO4 (FW 177.99) 12 g of KH2PO4 6 g of NaCl 30 g of Glucose (at least 30 g) Dissolve in 1L H20: 50 mL Basal medium eagle vitamins 400 uL of 10 mM FeCl3 400 uL of 10 mM ZnCl2 4 mL of 100 mM CaCl2 2 mL of 1 M MgSO4 Add the 3 L to 1 L of H20 and adjust pH to 7.6 with concentrated HCl (should start at around 9.5) Add 500 uL of H20 and 500 uL of 8N KOH to each of 4 L-SeMet bottles (100 mg each). Mix by shaking. Add to 4 L solution using pipet to ensure all liquid removed from bottles. Using sterile 0.2 uM filters, filter into sterile bottles. Next morning: Warm media to 37 C in water bath. Add 100 mg Amp per L. Save some media for OD blank. Add 2 mL of O/N per L. Grow until OD600 is 1.0 (at least 3-4 hours before starting to check). Induce with FRESH 1 mM IPTG for 5 hours at 37C. OD600 should be 1.7 to 1.9 upon harvesting cells. "#U^h  ! 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