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Team:Mexico-UNAM-IPN/Horizontal-Transfer/30 September 2008
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| - | #Culture | + | [[Team:Mexico-UNAM-IPN/Horizontal_Transfer/Implementation| <font face="trebuchet ms" style="color:Green"> '''Back to Implementation''' </font>]] |
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| + | #Culture overnight of JM101 and JM109 strains. | ||
#Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C. | #Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C. | ||
#Thaw on ice for for 10 minutes. transfer to 12 vials. | #Thaw on ice for for 10 minutes. transfer to 12 vials. | ||
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#Eliminate the supernatant and resuspend in 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Add 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Incubate 1hr at 4°C. | #Eliminate the supernatant and resuspend in 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Add 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Incubate 1hr at 4°C. | ||
#Transfer to 0.5ml eppendorf tubes and freeze. | #Transfer to 0.5ml eppendorf tubes and freeze. | ||
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| + | *<big>'''JM101'''</big>: In blue the corresponding vials to this ''Escherichia coli'' strain. | ||
Latest revision as of 03:42, 28 October 2008
- Culture overnight of JM101 and JM109 strains.
- Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C.
- Thaw on ice for for 10 minutes. transfer to 12 vials.
- Centrifugate for 7min at 4°C at 3500rpm. Eliminate supernatant and decant. fill the vials with the culture and centrifugate again. Repeat ≈ 3 times.
- Resuspend the pellet in 1ml of sterile cold 10mM MgCl2in each vial. Add 500µl of 10mM MgCl2 to eeach vial.
- Centrifugate at 3500 rpm for 7min at 40°C.
- Eliminate the supernatant and resuspend in 100µl of 50mM CaCl2, Glycerol at 20%. Add 100µl of 50mM CaCl2, Glycerol at 20%. Incubate 1hr at 4°C.
- Transfer to 0.5ml eppendorf tubes and freeze.
- JM101: In blue the corresponding vials to this Escherichia coli strain.
