IGEM:MIT/2007/Notebook/2007-6-14: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 21: | Line 21: | ||
Start: 2 vials of competent bacteria (500 uL each)<br> | Start: 2 vials of competent bacteria (500 uL each)<br> | ||
9 Transformations<br> | 9 Transformations<br> | ||
<pre> | |||
Contents Volume | |||
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) | |||
</pre><br><br> | |||
#Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs | #Aliquot 5 uL and 10 uL of ligation reactions into eppendorfs |
Revision as of 06:57, 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
Contents Volume 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