IGEM:MIT/2007/Notebook/2007-6-14: Difference between revisions
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#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs | #Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs | ||
#Pipette 50 uL of competent DH5a | #Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently | ||
#*Do not pipette up and down | |||
Revision as of 06:42, 14 June 2007
Grad Advisors
- Morning: Debbie
- Afternoon: Brian
LAB WORK
- Make competent E. coli cells
- Transformation into competent cells
- Plate transformed cells
Depending on how fast cells grow......
- Night time
- Analyze plates
- innoculate 4ml LB/Amp
Procedures
Regular cell membranes are not permeable to plasmids, so we much use ions (CaCl) to make the bacteria competent and able to accept foreign DNA. Start: 2 vials of competent bacteria (500 uL each) 9 Transformations U V (2 uL) dV (5 uL) dV + dI (5 uL) ddV (5 uL) ddV + ddI (5 uL) dV (10 uL) dV + dI (10 uL) ddV (10 uL) ddV + ddI (10 uL)
- Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs
- Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
- Do not pipette up and down
