IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-13: Difference between revisions
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==Future Experiment Planning== | ==Future Experiment Planning== | ||
We'll update this later tonight. Here's a photo of our whiteboard. | We'll update this later tonight. Here's a photo of our whiteboard. | ||
[[Image:Cyano e coli experimental design.JPG| | <br> | ||
[[Image:Cyano e coli experimental design.JPG|300px]] |
Revision as of 15:43, 13 July 2006
Incubator status
- Removed
- ...
Experimental Results
The plates grown from yesterday (2nd Topo&Transformation) show the following:
Positive Control: Many colonies
Negative Control: No colonies
Tranformed Plate: One in the middle, three+ on the edge of the plate.
DNA Miniprep of Topo+KaiABC plasmid
Using the regular Qiagen Protocol (Vs.2 of DNA miniprep booklet, p22) - DNA is stored in a 1.5uL blue marked microcentrifuge tube, 50uL dH20, 62.6ng/uL.
Liquid culture growth
Hetmann grew another liquid culture for use for glycerol stock.
Site-Specific Mutagenesis PCR
Refer to the image for details; this is the "crossover pcr" step.
For this step, we are going to create 4 PCR constructs with primers which will create the site mutation we want to eliminate the restriction sites.
The 4 sites are:
- kABC_3_9
- kABC_10_7
- kABC_8_5
- kABC_6_4
Where:
- 3=crossF
- 4=crossR
- 5=pst1R
- 6=pst1F
- 7=pst2R
- 8=pst2F
- 9=eco1R
- 10=eco1F
and:
- the kABC_x_y signifies the sequence bounded by primers 'x' and 'y'
The cycle for this PCR is:
*#95@15' *#94@30" *#56@30" *#72@3'30" *#Cycle to step 2, 40x *#72@5' *#4@forever
Each pot should have:
Using the Roche Vent PCR Kit | |
0.5 μL template | |
5 μL 10x buffer | |
1 μL 10 mM dNTP | |
1 μL Primer 1 | |
1 μL Primer 2 | |
0.5 μL Taq | |
41 μL dH20 | |
Future Experiment Planning
We'll update this later tonight. Here's a photo of our whiteboard.