Team:Michigan/Pili August September
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''Kevin, Marc, Alena'' | ''Kevin, Marc, Alena'' | ||
- | Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store | + | Alena order/picks up NEB Ligase (T4 DNA Ligase, 20,000U/mL) from MSRB II enzyme store. |
Attempted experiment: | Attempted experiment: | ||
*Added 1uL of CIP (Calf Intestine Phosphatase) to plasmid reactions only (pBAD) | *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 | **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 (65ºC 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>> [https://2010.igem.org/Team:Michigan/Pili_Expression#8.2F14.2F2010] 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.''' | '''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>> [https://2010.igem.org/Team:Michigan/Pili_Expression#8.2F14.2F2010] 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: | 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 4ºC fridge |
==8/17/2010== | ==8/17/2010== | ||
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Met with Chris to discuss about yeast agglutination | Met with Chris to discuss about yeast agglutination | ||
- | *Chris sent us a possible paper with a decent protocol to look at for the assay [[Media:YeastAgglutinationPaper.pdf]] | + | *Chris sent us a possible paper with a decent protocol to look at for the assay [[Media:YeastAgglutinationPaper.pdf]]. |
'''NOTE ABOUT K.O. STRAINS''' | '''NOTE ABOUT K.O. STRAINS''' |
Latest revision as of 03:05, 27 October 2010