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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

Practiced Gibson Assembly

Gibson Reaction Mix for All Samples

Mass (μg)	Enzyme (μL)	Buffer (μL)	Total (μL)
1	1	2	20

	Con (ng/μL)	Vol DNA (μL)	Vol H2O (μL)
1.1	151	6.62	10.38	CMU-Nde1
1.2	267	3.75	13.25
1.3	141	7.09	9.91
2.1	652	1.53	15.47	TRE-t-Sta1
2.2	263	3.80	13.20
2.3	212	4.72	12.28
4.1	149	6.71	10.29	TRE-t-Sta1
4.2	415	2.41	14.59
4.3	583	1.72	15.28
6.1	353	2.83	14.17	TRE-t-Sta1
6.2	593	1.69	15.31
6.3	361	2.77	14.23
7.1	448	2.23	14.77	Het1a-Xhol
7.2	237	4.22	12.78
7.3	186	5.38	11.62
8.1	246	4.07	12.93	Het1a-Lac-BamH1
8.2	182	5.49	11.51
8.3	871	1.15	15.85
9.1	410	2.44	14.56	TRE-t-Sta1
9.2	214	4.67	12.33
9.3	185	5.41	11.59
10.1	285	3.51	13.49	CMU-Nde1
10.2	547	1.83	15.17
10.3	388	2.58	14.42

PCR reaction to check assemblies; ran the products through two gels (one gel's purpose was to check the products' size by inspection under UV, the other gel's purpose was to extract the products)

Extracted plasmids from transformed bacteria (LR reaction); measured plasmid concentration with nanodrop