IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/23

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Team Allergy

Procedures

Today's objectives are two fold. One, we want to prepare RS300 bacteria containing an (unwanted) amiRNA such that it is ready to accept our amiRNA sequence. Two, transformation of E. coli with our prepared V0120 vectors containing arabadopsis allergens LTP sense, Ger 3 sense, and Ger 3 antisense.

  1. Growing RS300 Bacteria Colonies
  2. Transformation of Competent E. coli with V0120 containing allergens LTP sense, Ger 3 sense, and Ger 3 antisense

Beyond lab procedures, we also took time today to design the primers for use in amiRNA for allergens LTP sense, Ger 3 sense, and Ger 3 antisense. To construct the mini hairpin RNA, each allergen will need four primers in addition to the biobrick end primers to remove the existing amiRNA sequence.

We also took time today to design flowcharts for our amiRNA PCR reactions. There are two different ways we can perform the procedures: sewing all three constructs at once or sewing the first two and then the third. The former is less robust, but is easier because it requires fewer steps.

Results

1. Growing RS300 Bacteria Colonies

After these grow, we will extract the DNA from these bacteria and wait for the primers to arrive. Once they do, we will continue to replace the existing amiRNA with our own sequence.

2. Transformation of Competent E. coli with V0120 containing allergens LTP sense, Ger 3 sense, and Ger 3 antisense

We are still awaiting results. If the bacteria colonies grow, then they have taken up the plasmids. We will check for the correct sequences tomorrow.

Team Fence

Minipreps

The following colonies were minipreped after passing the night in the shaking incubator (37°C)

  • PMT413
  • PMT316
  • firefly luciferase
  • GFP (mut)
  • cre recombinase
  • lox66
  • lox71

Nanodrops for above minipreps

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

If two numbers are given, separated by a slash, then two measurements were taken, both are presented.


Plasmid Quantity (ng/μL) 260/280
Lox 71 #1 41.4 1.82
Lox 71 #2 59.6 1.89
Lox 71 #3 17.4/80.2 1.98
GFP #1 21.2 1.83
GFP #2 110.0 1.90
GFP #3 60.9/124 1.88
pMT316 #1 11.6 1.76
pMT316 #3 16.5 1.81
Cre #1 84.9 1.93
Cre #2 107.7 1.88
Lox66 #2 51.3 1.87
Firefly Luciferase #2 309.3 1.91
Firefly Luciferase #3 325 1.9
pMT413 #1 461.7 1.89
pMT413 #2 362.2 1.91
pMT413 #3 381.8 1.92




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