3/7/14
Made two LR reactions as practice.
First aliquot, 4 uL total:
- 1 uL of 5 fmol/uL tag-BFP
- 1 uL of 5 fmol/uL Hef1a promoter
- 1 uL of 10 fmol/uL Destination vector
- 1 uL of LR enzyme
Second aliquot, 4 uL total:
- 1 uL of 5 fmol/uL tag-BFP
- 1 uL of 5 fmol/uL TREt promoter
- 1 uL of 10 fmol/uL Destination vector
- 1 uL of LR enzyme
Stayed on the counter at room temperature overnight
3/8/14
Did not take notes today--need to ask Kyle for more details on protocol
Transformed the LR reactions from 3/7/14 into bacteria
- Added 1.5 uL of LR reaction into bacteria in a 1.5 mL eppendorf tube
- Let the bacteria incubate on ice for ~30 minutes
- Added SOC medium to the bacteria
- Heat-shocked the bacteria for 30 seconds, then sat them on ice for 2 minutes
- Incubated the bacteria (50 C?) in the rolliing-incubator for 1 hour (?)
- Plated the bacteria on LB-Amp plate
- Let the LB-Amp-plated bacteria incubate until the next lab day
Prepared a PCR reaction for practice
- The segment to be amplified is the right side of a gene coding for YFP (other people are amplifying the left side)
- The goal will be to amplify the right and left sides of the gene and then assemble them together into a functional gene
- Diluted the two primer solutions (forward primer and reverse)
- Added pre-made "PCR Mix" and primers to DNA
- Set PCR machine to the cycle specifications called for in the protocol
3/14/14 & 3/16/14