Good thing takes time

Medium:
SO (1 liter) : tyrptone 20g
yeast extract 5g
NaCl 0.5g
KCl 2.5mM adjust pH to 7.0 (0.2ml NaOH 5M)
autoclave

SOB: add MgCl2 10mM; MgSO4 10mM

Fresh prepare:
RF1: RbCl 100mM
MnCl2.4H2O 50mM
KoAc 30mM
CaCl2.2H2O 10mM
Glycerol 15% adjust pH to 5.8 with 0.2M acetic acid
sterile by 0.2u filter

RF2: MOPS 10mM
RbCl 10mM
CaCl2.2H2O 7.5mM
Glycerol 15% adjust pH to 6.8 with NaOH
Sterile by 0.22 filter

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Working Medium//
125mL SO :

trypton 2.5g
yeast extract 0.025g
NaCl 0.0625g
1M KCl 312.5uL
ddH2O 125mL adjust pH to 7.0 with NaOH by testing paper//Autoclave

add 1M MaCl2 1.25mL/ 1M MgSO4 1.25mL

Fresh prepare RF1 & RF2 then in 4oC
*50mL RF1
RbCl 0.6g
MnCl2.4H2O 0.495g
KoAc 0.147g
1M CaCl2 0.5ml
50% Glycerol 15mL
ddH2O 50mL ..adjust pH to 5.8 with acetic acid by testing paper// 0.22 u filter

*10mL RF2
RbCl 0.12g
1M MOPS 0.1 ml
1M CaCl2 75uL
50% Glycerol 3ml
ddH2O 10ml .. adjust pH to 6.8 by paper// 0.22 u filter


*Competent cell
Day I: prepare fresh plate
Day II: inoculate 2-5ml SOB with 2-3 colony, 37oC overnight
Day III: inoculate o/n culture (1:100) to 125ml SOB, 270rpm. to O.D.550=0.2-0.3 (5E7cell/ml)
//following steps should be on ice//
pellet cells at 2.5k, 4oC 12-15min;
Resuspend cell in 1/3 volumn RF1, place on ice for 15 min to 2 hrs, pelet at 2.5k, 4oC 12-15min;
Resuspend cell in 1/12.5 ml volumn RF2, swirling on ice for 15min;
Aliquote 100-200ul/tube (pre-chilled on ice);
store at -70oC

Efficiency test by Fu-Kai is about 3E6 cells/ug...