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 | ||
#*For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/uL) | |||
#Pipette 50 uL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently | #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 | #*Do not pipette up and down | ||
#Incubate on ice for 60 minutes | #Incubate on ice for 60 minutes | ||
#Heat shock the bacteria in a 37C water bath for 30 seconds | #Heat shock the bacteria in a 37C water bath for 30 seconds |
Revision as of 06:53, 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
- For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/uL)
- 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
- Incubate on ice for 60 minutes
- Heat shock the bacteria in a 37C water bath for 30 seconds