Team:Michigan/Pili Expression
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#use 5 PCR reaction tubes for each; 5 for fimB and 5 for pBAD | #use 5 PCR reaction tubes for each; 5 for fimB and 5 for pBAD | ||
#add the following amounts in that order (total volume of 20uL) | #add the following amounts in that order (total volume of 20uL) | ||
- | + | *16 uL of the DNA (fimB and pBAD to their respective tubes) | |
- | + | *2 uL of NEB 2 buffer | |
- | + | *1 uL NcoI | |
- | + | *1uL HindIII | |
#incubate for 37C overnight (12hrs) | #incubate for 37C overnight (12hrs) | ||
##place into 4C fridge the next day | ##place into 4C fridge the next day | ||
+ | |||
+ | ==8/16/2010== | ||
+ | ''Kevin, Marc, Alena'' | ||
+ | |||
+ | Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store | ||
+ | |||
+ | Attempted experiment: | ||
+ | *Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD) | ||
+ | **CIP will cut off the 3' phosphate to prevent the plasmid from folding back on itself | ||
+ | *incubate CIP-pBAD at 37C for 1 hr and then heat-shock (65C for 15 min) | ||
+ | We made a huge human error when setting up our thermal cycle program on the PCR machine. We did not realize we only set the thermal cycle for our digest on 8/14/2010 for 12 minutes (12:00) rather than the intended time period of 12 hours (12:00:00). This explains the condensation present in the PCR machine (it stayed at 4C for too long). We discovered our mistake after running the incubation of CIP+pBAD for 37C for (1:00) 1 minute then heat-shock at 65C for 15 minute. It is VERY IMPORTANT to triple check one another when entering in any program. | ||
+ | |||
+ | *Rest of the day: | ||
+ | **headed over to the ERB and made two cultures of 5mL LB-Amp (in 50mL conical tubes)--> placed in the Lin Lab 4C fridge | ||
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Revision as of 04:54, 17 August 2010