IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-13

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(2006-07-13)

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.

Refer to this for PCR overview

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:

• 1. kABC_3_9
  2. kABC_10_7
  3. kABC_8_5
  4. 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
1 μL template
5 μL 10x buffer
1 μL 10 mM dNTP
1 μL Primer 1
1 μL Primer 2
0.5 μL Taq
40.5 μL dH20

Future Experiment Planning

More detailed plans for our future cyanobacteria work: