Difference between revisions of "Team:RPI Troy NY/Description"

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Project Description

♦Background♦

♦Design♦

In this project, we sought to increase the production of sophorolipids by taking a two-part approach. In the first part, we engineered primers and inserted these into the yeast to be able to produce either normal amounts of sophorolipids or lesser amounts of sophorolipids (gene knockout). In the second part, we extracted these sophorolipids and completed analysis on this.

Protocols

♦Microbial Genetics Protocols♦

Agarose Gels

Analytical Gel

• 1-2% Agarose

• Ethidium Bromide (1uL/100mL)

Purification Gel

• .8% Agarose

• Ethidium Bromide (1uL/100mL)

PCR Amplification

• 50ng Template DNA

• 25uL Accuzyme or Hotstart Mix (1uL/100mL)

• 1uL primer stock (10mmol forward and reverse primer)

• Remainder MilliQ
1uL pure DMSO (Optional, use if high GC content)

50uL total

Fusion PCR

• 100ng each PCR amplicon (equimolar value)

• 25uL Accuzyme or Hotstart Mix (1uL/100mL)

• Remainder MilliQ

50uL total

Run 10 cycles of appropriate annealing/joining protocol in Thermalcycler

immediately add 1uL primer stock (10mmol forward and reverse primer)
Run 20 cycles of appropriate annealing amplification and extension protocol in Thermalcycler
We grew the yeast in two types of broth: FM2 and PM2. This is the components of each of those broths.

PM2 Ingredients Amt.(g/L) FM2 Ingredients Amt.(g/L)

Glucose 200 Glucose 100

Yeast Extract 5 Yeast Extract 5

KH2PO4 1 KH2PO4 1

MgSO4 0.5 MgSO4 0.5

CaCl2 0.1 CaCl2 0.1

NaCl 0.1 NaCl 0.1

Peptone 0.7 Peptone 0.7

Experiments

Notebook

@@ Line 107: / Line 107: @@
 <p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">• 50ng Template DNA</p>
 <p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">• 25uL Accuzyme or Hotstart Mix (1uL/100mL)</p>
-<p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">• 1uL pure DMSO (Optional, use if high GC content) </p>
 <p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">• 1uL primer stock (10mmol forward and reverse primer)</p>
 <p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">• Remainder MilliQ </p>
+<i><p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3">1uL pure DMSO (Optional, use if high GC content)</p></i>
 <p style="padding-left:25px"><font face= "Times New Roman"> <font size ="3"> 50uL total </p>
 <p></p>