User Manual
Contents
1. lt gt MCLAB Molecular Cloning Laboratories User Manual Version 1 0 Revision Date 07 30 2013 Product name BL21 DE3 Competent E Coli Cat BS 100 BS 196 Description High yields high efficiency no heat shock necessary Recommended Storage Conditions This product should be stored at 80 C Thaw on ice only before use Do not re freeze Transformation Steps 1 Thaw one vial of cells on ice per transformation 2 Add 5 10 ng of DNA in a volume of 1 5 uL to the cells and mix by tapping gently Do not mix cells by pipetting 3 Incubate the vial s on ice for 30 minutes 4 Heat shock the cells by incubating the vial s for exactly 30 seconds in the 42 C water bath Do not mix or shake 5 Remove the vial s from the 42 C bath and quickly place on ice 6 Add 250 uL of pre warmed SOC medium to the vial s SOC is a rich medium use proper sterile technique to avoid contamination 7 Secure the vial s in a microcentrifuge rack with tape Place the rack in a shaking incubator and shake the vial s at 37 C for 1 hour at 225 rpm 8 Plate two different volumes of the transformation reaction onto LB plates containing the appropriate anti biotic for plasmid selection Include 34 g mL chloramphenicol if using BL21 DE3 pLysS or BL21 DE3 pLysE cells Select two volumes ranging from 20 200 uL to ensure well spaced colonies on at least one plate The remaining transformation reaction may be stored at 4 C and plat2. e transformation mixture to select for individual clones See next page for details 2 lt MCLAB
3. ed out the next day if needed 9 Invert the plates and incubate at 37 C overnight 10 Select transformants from the plates and culture as described on page 8 Note Clones may exhibit differences in expression of heterologous genes We recommend choosing 3 4 1 650 872 0245 www mclab com 1 transformants when characterizing clones for protein expression Expression Steps 1 Pick 3 4 transformants for overnight culture in 5 mL LB medium containing antibiotic to select for your expression plasmid If using BL21 DE3 pLysS or BL21 DE3 pLysE add 34 g mL chloramphenicol to select for pLysS or pLysE Grow overnight at 37 C with shaking to saturation OD600 gt 22 2 Use the overnight cultures to inoculate fresh LB medium containing antibiotic to an OD600 of 0 05 0 1 1 50 dilution of the overnight culture This dilution allows the cells to quickly return to logarithmic growth and reach the appropriate cell density Use a volume appropriate for taking time points if desired Note If you are using BL21 DE3 pLysS or BL21 DE3 pLysE you may choose not to include chloramphenicol in these cultures Generally the cells will not lose the pLysS or pLysE plasmid during the limited number of cell doublings that occur in the growth and induction stages 3 Use the remainder of each overnight culture to create glycerol stocks Once you have identified the clone that best expresses your protein you can use the glycerol stock to perform additional ex
4. pression experiments 4 Grow the cultures until they reach mid log phase OD600 0 4 2 to 3 hours 5 Induce the cultures by adding IPTG to a final concentration of 0 5 mM and culture for an additional 2 3 hours You may also take time points to analyze for optimal expression of your protein 6 Analyze clones by western blot or enzymatic assay to determine which clone best expresses your protein of interest Use the glycerol stock created from this clone for expression experiments If you find that expression levels in subsequent inductions decrease or you find that you lose your plasmid your protein may be toxic to E coli Expression Guidelines 1 Use BL21 DE3 pLysS or BL21 DE3 pLysE strains for expression experiments Both strains produce T7 lysozyme to inhibit the action of T7 RNA polymerase and reduce basal level expression of the gene of inter est Choose BL21 DE3 pLysE when the tightest regulation of expression is required 2 Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase i e DH5a JM109 etc 3 Perform a fresh transformation of BL21 DE3 pLysS or BL21 DE3 pLysE cells before each induction experi ment 4 Minimize the amount of time that the cells bearing the gene of interest are cultured before IPTG induction 5 Following transformation of BL21 DE3 pLysS or BL21 DE3 pLysE cells grow cells in SOC medium for 1 hour and go directly to protein expression Do not plate th
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