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):

LaneSegmentPerson
leftmost laneladdern/a
1AdapterKL
2Q1-Q9AS
3Q9-QXAL
4?KL
5?JA

 

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

 

 

 

 

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