ࡱ> BDA5@ ("bjbj22 $$XXC zzzzzzz,3LLLLLLLL$R;9zLLLLLzzLLLLLLvzLzLLLLLfzzfL@ OM9:f03fFfzzzzzf(LLLLD$B Competent cell preparation using rubidium chloride This procedure is from the Promega Protocols and Applications Guide (3rd edition), p. 45-46. 1. Inoculate a single colony from an LB plate in 2.5 ml of LB medium. Incubate overnight at 37C with shaking (approximately 225 rpm). 2. On the following day, use the entire overnight culture to inoculate 250 ml of LB medium containing 20 mM MgSO4 (this results in a 1:100 dilution). Grow the cells in a 1L baffled flask until the A600 reaches 0.40.6 (typically 23 hours). A 1L flask is necessary for proper aeration during growth. 3. Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4C. For a 250 ml culture, use two 250 ml centrifuge bottles in a large rotor (Sorvall GSA, Beckman JA-14). 4. Gently resuspend the cell pellets in 0.4 volume (based on the original culture volume) of ice-cold TFB1. For a 250 ml subculture, use 100 ml TFB1 (50 ml/bottle). Combine the resuspended cells in one bottle. For the remaining steps, keep the cells on ice and chill all pipets, tubes and flasks. 5. Incubate the resuspended cells on ice for 5 minutes. 6. Pellet the cells by centrifugation at 4,500 x g for 5 minutes at 4C. 7. Gently resuspend the cells in 1/25 of the original culture volume of ice-cold TFB2. For a 250 ml subculture, use 10 ml of TFB2. Note: Treat the competent cells gently as they are highly sensitive to handling and elevated temperature. 8. Incubate the cells on ice for 1560 minutes and then aliquot into prechilled tubes. Quick-freeze the tubes in liquid N2 and store at -70C. TFB1 250ml 30 mM potassium acetate 0.74g 10 mM CaCl2 1.25ml of 2M 50 mM MnCl2 2.47g 100 mM RbCl 3.023g 15% glycerol 37.5ml Adjust pH to 5.8 with 1M acetic acid (glacial acetic acid ~17.4N). Filter-sterilize (0.2 mm). Note: take care when titrating this solution; if you overshoot and try to bring the pH back up with hydroxide, the manganese will fall out of solution. TFB2 100ml 10 mM MOPS or PIPES 0.335g 75 mM CaCl2 3.75ml of 2M 10 mM RbCl 0.12g 15% glycerol 15ml Adjust pH to 6.5 with 1M KOH. Filter-sterilize (0.2 mm). TFB1 and TFB2 can be stored at room temp or 4C. They should be ice-cold for the procedure. Additional notes 1. The cells do not resuspend very easily in TFB1. Be patient and b4Xx{} | } 3 8 26;=@AY`lmp} >FZ^þȺ׵׵ȺȺȜȺ׵ȺȺh_h_OJQJ h$bh_ h$bh$b h$bH*h$b hAC5 h$b5 h#6h_h#6h_5h#6h_H*h#6h_H*h#6h_6h_h_h_5CJaJ=45  J K ) * b c 1 2 12Aa}&&&&&&&&&&&&&&&&&&&&&&&&&&& ^`gd_$a$gd_("<>\ RTvx!!!!""("&&&w&&&&&&&&Q&&&`gd_gd_ ^`gd_ !!!!" "("h_h_H*h_U h#6h_h_h_OJQJ e gentle (pipet up and down, and swirl the bottledo not vortex). I have never achieved a uniform suspension; there are always some small clumps of cells present. Though the procedure does not specifically say so, I keep the bottles on ice while I am resuspending in TFB1. 2. The cells resuspend much more easily in TFB2. I usually let them sit for 45 min to 1 hour before aliquoting. 3. I usually get excellent competency using this procedure (1.5x107), and I have used the cells for up to a year afterwards. 4. Heat shock for 45 sec at 42C. ":p_/ =!"#$%<@< NormalCJaJmH sH tH DA@D Default Paragraph FontRiR  Table Normal4 l4a (k@(No ListJ%@J uEnvelope ReturnCJOJQJaJC $C 45JK)*bc1212Aa}*+fg. /   E 000000000 0000 000000 00000 00 0 00 00 0 00 00(0 0 0 0 0000000000045JK)*bc1212Aa*+fg. /   E 0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0̀0000000000000000000000000000008(" (" (" EGeg E 6?JdE 33332B +JgE E svetal99i$bAC_@B B ALLB B 4 C P@PPP@@UnknownGz Times New Roman5Symbol3& z Arial qhj쒩&   24d> > @ <83Competent cell preparation using rubidium chloridesvetal99Oh+'07 ( D P \hpx4Competent cell preparation using rubidium chlorideompompompNormaln svetal99 ce10tMicrosoft Word 10.0@@Z _@`bB9 G5VT$m    ."System+r0( -@Times New Roman- 22 Competent cell preparationT;`A4'4A'44 !A44@;3;' ;@/2 :  using rubidium chlorideA.!@;4A@!A A`4A!;4!A4 2  >@Times New Roman- 2  --@2 #This procedure is from the Promega =2'2!2,,22",'!!2N2,8!2N,2,@Times New Roman-;2 b Protocols and Applications Guide='22,2'223=22,222'H22,-2   (3!2@Times New Roman-2 Q#rd"-#2 [ edition), p. 45,222!222 2 -!2 46.22 2 C - 2  -2 e1.2 2 e+ &2 eQ_Inoculate a single colony from an LB plate in 2.5 ml of LB medium. Incubate overnight at 37C 23,2,,,'31,,2241!!2N,2<C2,,222N2!<BN,22N 2,23,,22,!212,22(C2 Q with shakinH2'2,2222 !g (approximately 225 rpm).1",22!23N,,0222!2N! 2 q . 2 K -2 2.2 2 + &2 QcOn the following day, use the entire overnight culture to inoculate 250 ml of LB medium containing H22,!22H213.02(,2,,2!,23,!212,22!,222,2,,222N2!<BN,22N,22,231-2 1Q 20 mM MgSO22NYY18H- 2 >4"-2 1V (this results in a 1:100 dilution). Grow the cells in a 1L baffled flask until the A!2'!,'2'2,22222222!H!2H2,-,'2,3;3-!!,2!,'2222,H-2 >W600"""- 2 1 2 Q reaches 0.4!,,,3,'22 2 22 D 0.6 (typicall22!02,,2 (y 202 2 2s2 E3 hours). A 1L flask is necessary for proper aeration during growth.2222!(!H3<!,'3'2,,,'',#0!2!2!23,!-,!,2222!311!2H2 2  . 2  --2 3.2 2 + &P2 Q.Pellet the cells by centrifugation at 4,500 x 8,,2,,,'30,,2!"21,22,22223- 2  g2-\2 P 6 for 5 minutes at 4C. For a 250 ml culture, use two !2!3N22,',2(C72!,322N,22!,2',H2p2 QC250 ml centrifuge bottles in a large rotor (Sorvall GSA, Beckman JAh222N,,2!!31,22,(2,,"1,!22!!82!2,H8HB-,2N,2(H 2  -!2 6 14).22! 2   - 2 p -2 4.2 2 + &2 Q]Gently resuspend the cell pellets in 0.4 volume (based on the original culture volume) of iceH,20",'2'2,222,,,2,,'222222N,!2-',2222,2!12,,22",222N,!2!-, 2 -!2 cold o,222 VQbTFB1. For a 250 ml subculture, use 100 ml TFB1 (50 ml/bottle). Combine the resuspended cells in =7B272!,222N'22,22!,2',222N=8C2!22N22,!C2N22,2,!,'2'2-22,2-,'2d2 Q;one bottle. For the remaining steps, keep the cells on ice22,22,72!2,!,N,221',2'2-,22,-,'22,,G2 3 ( and chill all pipets, tubes and flasks.,22,2,22,(22,',22!,'2' 2  . 2 <  -2 5.2 2 + &Y2 Q4Incubate the resuspended cells on ice for 5 minutes. 3,22,,2,!,'2'2,23,3,,'22,,!2!2N22-' 2 f  - 2 "  -2 6.2 2 + &s2 QEPellet the cells by centrifugation at 4,500 x g for 5 minutes at 4C.8,,2,,,'30,,2!"21,22,222231!3!3N22,',2(C 2  - 2   -2 { 7.2 2 { + &w2 { QHGently resuspend the cells in 1/25 of the original culture volume of iceH,20",'2'2,222,,,'22222!2,2!12,-22!,222N,2!,, 2 { [ -"2 { } cold TF ,23=7&2 { B2. For a 250 ml B272!,222N82 Qsubculture, use 10 ml of TFB2.'22,22!,2',22N2!=7B2 2 / . 2 a  -- 2  q@Times New Roman-2 QNote:TH2!,!-2 9Y Treat the competent cells gently as they are highly sensitive to handling and elevated >!,,2,,2N2,-2,,'1,20,'3.0,"-2120',2'2,22,2221,22,,2,,22 G Q temperature.,N2,!,2", 2 G G .- 2  6-2 -8.2 2 -+ &;2 -Q Incubate the cells on ice for 15 3,22,,2,,,'22,,!2!22 2 -!2a2 -S960 minutes and then aliquot into prechilled tubes. Quick22N22,',222,2,222222!,,2,322,'H2,2 2 -3-!2 -Tfre!",2 -eze the ,-,2,-%2 Qtubes in liquid N22,'2222H- 2 2"- 2  and store at ,22'1!,, 2 -!2 /70C. 22(C 2 I . 2  --2 TFB1C<C3 2  7 2   , 2 8 , 2 d , 2  ,2 25222  0ml2R 2  7- 2  q.2 Q30 mM potassium acetate22NY22,''2N,,,,, 2 Q ? 2  ,2 0.74g 2221 2  .- 2 l q2 lQ 10 mM CaCl22NYC,C- 2 y\2!- 2 y} - 2 yd ,- 2 y ,-2 l 1.25ml of 2M222N2!2Y 2 l  -- 2  q2 Q 50 mM MnCl22NYY2C- 2 w2!- 2  - 2 d ,- 2  ,-2 2.47g 2221 2  . 2 R q2 RQ 100 mM RbCl222NYC2C 2 R 2 Rd , 2 R ,2 R3.023g22221 2 R . 2  q2 Q 15% glycerol22S10-,!2 2  2 d , 2  ,2 37.5ml222N 2  - 2 8 --A2 $Adjust pH to 5.8 with 1M acetic acidH22'2H222H22Y,,,,-,242  (glacial acetic acid ~17.4"1,,,,--,,,262222  N)H!2  . Filter7,! 2 = -!"2 ^ sterilize (0.2 (,!-,!22@Symbol- 2 mm8-2 m). N!-2 FNoteT;'4- 2 0:!-2 Q 2 1itake care when titrating this solution; if you overshoot and try to bring the pH back up with hydroxide, ,2,,-!,H3,2!,212''2222!/2222-!(222,22"022!312,2H2,,222H2402!232,G2 (the manganese will fall out of solution.2,N,22,2,',H!,222!'2222 2  , 2  --2 TFB2C<C3 2  7 2   , 2 8 , 2 d , 2  ,2 100mll222R 2  7- 2  q-(2 Q10 mM MOPS or PIPES22NYYH882!88=9 2 ) g 2  ,2 0.335g22221 2  .- 2 p q2 pQ 75 mM CaCl22NYC,C- 2 }\2!- 2 }} - 2 }d ,- 2 } ,-2 p 3.75ml of 2M222N2!2Y 2 p  -2 Q 10 mM RbCl22NYC2C 2 a  2 d , 2  ,2 0.12gg2221 2  . 2 V q2 VQ15% gg22S12 VRlycerol 0-,!2 2 Vi 2 Vd , 2 V ,2 V15ml22N 2 V - 2  --C2 D%Adjust pH to 6.5 with 1M KOH. FilterH22'2H222H22YHHH7," 2 D-!"2 D sterilize (0.2 ',!-,!22- 2 D. m8-2 Df m). N! 2 D  . 2  -q2 )DTFB1 and TFB2 can be stored at room temp or 4C. They should be ice=7B2,22>7B2,,22,(2!,2,!22N,N22!2)C=2-0'22222,-, 2 ). -".2 )P cold for the procedure.-22!2!2,2!2-,22!, 2 ) .-                    ՜.+,0  hp  -CH> A 4Competent cell preparation using rubidium chloride Title  !"#$%&'()*+,-./012345678:;<=>?@CRoot Entry F0kM9E1TableWordDocument$$SummaryInformation(H7DocumentSummaryInformation89CompObjj  FMicrosoft Word Document MSWordDocWord.Document.89q