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