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From 2012.igem.org

Contents 1 I. Meeting with Kaury Kucera about primer designs: 2 II. Resuspended all remaining primers in 200 microliters sterile DIW and found their concentrations using a spectrophotometer 3 III. Ran PCR (did all appropriate calculations and dilutions for reaction) with primers 5 and 6, using template RiG DNA 3 using the following protocol: 4 IV. Observed, recorded, and photographed results of the freezing in salt solutions (Red) experiment: 5 V. Started Green Experiment

I. Meeting with Kaury Kucera about primer designs:

• Confirmed findings about primer design for site-directed mutagenesis as follows:

1. Mutations should be made closer to 3’ end
2. G’s are desirable at 5” ends
3. G’s and C’s are desirable at 3’ end, but not so that one nucleotide repeats 3 or more times
4. TM should be 10 degrees higher than annealing temperature (at least 65 when annealing temperature is set at 55)
5. 6 - 10 nucleotides at 5’ ends of primers and 10-20 at 3’ depending on GC/TA content due to H-bonds.
6. To knock out columns of T’s, substitute with alanine or serine to ensure formation of H-bonds and stable secondary structure
7. Dimerization is possible with substitution or insertion at or near turns, while conserving G’s
8. 40-45 is the max base-pair length

II. Resuspended all remaining primers in 200 microliters sterile DIW and found their concentrations using a spectrophotometer

III. Ran PCR (did all appropriate calculations and dilutions for reaction) with primers 5 and 6, using template RiG DNA 3 using the following protocol:

1. Mix the following reagents in this order:

1. x sterile, pure DIW so that total volume adds to 50 microliters (20 microliters)
2. 10 microliters 5x Phusion HF Buffer for a final concentration of 1x (4 microliters)
3. 1 microliter 10mM dNTP mix for a final [200micromolar each] (0.4 microliters)
4. 1 microliter 25 micromolar primer A for a final [0.5 micromolar]
5. 1 microliter 25 micromolar primer B for a final [0.5 micromolar]
6. 0.5 micrograms template DNA (.2 micrograms) = 500 ng/[from spectrophotometer in ng/microliter] = amount to be added
7. 1.5 microliters DMSO for a final 3% (0.6 microliters)
8. 0.5 microliters Phusion DNA Polymerase (0.2 microliters)

IV. Observed, recorded, and photographed results of the freezing in salt solutions (Red) experiment:

Andriana Notebook 4.jpg

Andriana Notebook 5.jpg

Andriana Notebook 6.jpg

Plate	Properties
1 A	Plate completely covered
3 A	Plate completely covered
1 B	~900 colonies
3 B	~150 colonies
1 C	~5000 colonies
3 C	~400 colonies
1 D	~1000 colonies
3 D	Plate completely covered
1 E	clear
3 E	clear
1 F	~2000 colonies
3 F	~800 colonies

• Analysis of the above results:

• E. coli die in strong base solutions
• Pure water protects E. coli from ice damage the best
• 0.1 M NaCl causes greater E. coli mortality than 1M and especially 5M NaCl
• CaCl might be as effective as NaCl at increasing E. coli mortality, but at a different concentration if so
• Contrary to the expected result, transformed BL21 cells had lower survival across all tests. This might be do to higher sensitivity to preparation procedures/protocol, ironically due to adverse effect of expression of proteins from RiG plasmid, or due to the loss of plasmid that contains antibiotic resistance genes.

• New Questions:

• Why decreased survival in ionic solutions, especially in dilute NaCl?
• Will the above trends be observed if the experiment is repeated without freezing?
• Will the survival between transformed and normal origami cells vary also?

V. Started Green Experiment

1. Created A-E solutions to test cell survival when just suspended in them, rather than frozen

• A. 0.1M NaCl – 4 microliters 5M NaCl to 196 sterile, DIW
• B. 0.01M NaCl - .4 microliters 5M NaCl to 199.6 sterile, DIW
• C. 0.005M NaCl - .2 microliters 5M NaCl to 199.8 sterile, DIW
• D. 0.005M KOH - .2 microliters 5M KOH to 199.8 sterile, DIW
• E. 0.1M MgCl (stock present)

2. Although this change of condition might make the green experiment irrelevant to the red one, cell sample 2 and 4, untransformed but previously competent origami cells (controls from the transformation as “1” cells in relation to 3 were) and origami cells transformed with RiG plasmid respectively, were diluted 1:9 with sterile DIW from stock kept at 4C. From these solutions, 0.377 of cell type 2 and 0.256 microliters of cell type 4 were added to the solutions A-E for an estimated total of 10^3 cells.
3. The solutions were mixed via vortexing for a few seconds and allowed to sit at room temperature for about 30 minutes.
4. The entire volumes of cell solutions were plated on plain agar plates and incubated overnight at 37C.