User Manual
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1. lt gt MCLAB Molecular Cloning Laboratories User Manual Version 1 0 Revision Date 09 19 2013 Product name HB101 Competent E Coli Cat HB 100 HB 196 Description High stability high efficiency no heat shock necessary For Research Use Only Application e deal for sub cloning and scale up applications e HB101 strain is a hybrid K12 x B bacterium containing the recA13 mutation that minimizes recombina tion and aidsin insert stability In addition it carries the hsdS20 rB mB restriction minus genotype which prevents cleavage of cloned DNA by endogenous restriction enzymes HB101 strain does not support Alpha Complementation for blue white screening Transform efficiency is around 1 0x107 1 0x109 cfu ug with pUC18 control DNA Recommended storage condition This product should be stored at 80 C Thaw on ice only before use Do not re freeze Recommended reaction conditions e Mix DNA with competent cell e Let stand on bench for 5 minutes e Direct plate Genotype F mcrB mrr hsdS20 rB mB recA13 leuB ara 14 proA2 lacY1 galK2 xyl 5 mtl 1 rpsL20 SmR glnV44 Protocol A stock pUC19 solution 0 1 ug ml is provided as a control to determine the transformation efficiency To ob tain maximum efficiency the experimental DNA must be free of phenol ethanol protein and detergents 1 Thaw competent cells on wet ice Place required number of autoclaved 1 5 ml microcentrifuge tubes on wet ice 2 Gen2. tly mix cells then aliquot 50 pl of competent cells into chilled microcentrifuge tubes If desired a 100 ul transformation may be performed 3 Refreeze any unused cells in the dry ice ethanol bath for 5 minutes before returning them to the 70 C freezer Do not use liquid nitrogen 4 To determine transformation efficiency add 5 ul 500 pg control pUC19 to one tube containing 50 ul com petent cells Move the pipette through the cells while dispensing Gently tap the tube to mix 5 Add 1 3 ul 1 10 ng of DNA of the DNA ligation reaction directly to a second tube containing 50 ul compe tent cells moving the pipet through the cells while dispensing Gently tap the tube to mix 6 Incubate the cells on ice for 30 minutes 7 Heat shock the cells for 20 seconds at 37 C If a 100 ul transformation is used heat shock for 45 seconds 1 650 872 0245 www mclab com 1 Do not shake 8 Place on ice for 2 minutes 9 Add 0 95 ml room temperature LB YT 1 or S O C Medium 10 Shake at 225 rom for 1 hour at 37 C for expression 13 x 100 mm glass tubes will hold microcentrifuge tubes in a shaker rack 11 After expression dilute 0 1 ml of the reaction containing the control pUC19 into 0 9 ml medium Spread 100 ul of the undiluted reaction and the 1 10 dilution on LB or YT plates with 100 ug ml ampicillin Vortex briefly before dilution in case cells have settled out Notes 1 Subcloning HB101 Competent Cells may be refrozen Subseq
3. uent freeze thaw cycles will reduce transfor mation efficiency approximately 2 fold 2 Do not use EDTA to stop ligation reactions Instead freeze ligations to store for later use The combination of DNA ligase and ligase buffer can inhibit transformation Optimal results are obtained with T4 Ligase and 5X ligase reaction buffer If desired ligations can be concentrated by ethanol precipitation prior to trans formation Dissolve dry pellet in filter sterilized TE buffer tRNA can be used as a carrier 3 Subcloning HB101 Competent Cells can support the replication of M13mp vectors However HB101 is F and cannot support plaque formation The competent cells should be added to top agar after lawn cells IPTG and Bluo gal or X gal have been added Incubation at 37 C for 1 hour is not required after addition of S O C Medium 2 lt MCLAB
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