Quint Lab:e.coli transformation chemical: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Marcel Quint (talk | contribs) No edit summary |
Marcel Quint (talk | contribs) |
||
Line 14: | Line 14: | ||
#Thaw on ice 100-200 µl of chemically competent cells. | #Thaw on ice 100-200 µl of chemically competent cells. | ||
#Briefly centrifuge the ligation reaction and place on ice. | #Briefly centrifuge the ligation reaction and place on ice. | ||
#Pipet 1-5 µl of the ligation reaction directly (if possible) into the competent cells (100-200 µl) and mix by tapping gently. Do not mix by pipetting! Store the remaining ligation reaction at –20ºC. | #Pipet 1-5 µl of the ligation reaction directly (if possible) into the competent cells (100-200 µl) and mix by tapping gently. Do not mix by pipetting! Store the remaining ligation reaction at –20ºC. | ||
#Incubate on ice for 30’. | #Incubate on ice for 30’. | ||
#Heat shock for 45’’ in 42ºC water bath. Do not mix or shake! | #Heat shock for 45’’ in 42ºC water bath. Do not mix or shake! | ||
#Place on ice for 1’, then add 1200 µl RT SOC or LB medium. | #Place on ice for 1’, then add 1200 µl RT SOC or LB medium. | ||
#Shake horizontally at 37ºC for 1 h at 200-225 RPM. | #Shake horizontally at 37ºC for 1 h at 200-225 RPM. | ||
#Spread 100 µl directly onto LB plate, then pellet cells, decant supernatant, resuspend pellet in remaining 100 µl SOC and plate. | #Spread 100 µl directly onto LB plate, then pellet cells, decant supernatant, resuspend pellet in remaining 100 µl SOC and plate. | ||
#Invert the plates and incubate at 37ºC O/N. | #Invert the plates and incubate at 37ºC O/N. | ||
#Select colonies and analyze by plasmid isolation, PCR, or sequencing. | #Select colonies and analyze by plasmid isolation, PCR, or sequencing. |
Latest revision as of 01:53, 26 July 2010
chemical transformation of e.coliBefore starting:
Procedure:
|