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Wisconsin-Madison/15 June 2009
From 2009.igem.org
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June 15, 2009
Ex 13: Speced Choline, ProU, YhfR, NudF from PCR Product and DNA is in very high conc.
Ran Gel: Only have Primers UNSUCCESSFUL PCR (rerun in 2 different procedures)
4uL of NTP’s instead of 1uL
* PCR reaction tubes are in a draw on the island
Re-Run #1: Phussion – 1.5 hr 50uL rxn
1uL Forward Primer
1uL Reverse Primer
0.5uL DNA Template
0.5uL DNA Polymerase
1uL 10mM dNTPs (in box in -20 that looks like tip box – use one vial
10uL 5x Phusion HF Buffer
Re-Run #2: Taq – 2.5 hr. 50uL rxn
1uL Forward Primer
1uL Reverse Primer
2uL DNA Template
21uL water
25uL Taq Master Mix
Ran Gels: 2.0% Agarose, 100bp Ladder, 25min, 90V
Phus(ProU, YhfR, NudF, Choline) L, Taq(Choline, ProU, YhfR, NudF), Temp(ProU, YhfR, NudF, Choline), Blank(14,15)
Results: PCR2: taq and Phussion are successes, single bands of products.
Ex 9: miniprep of Choline gene ran gel
Ex 11: Check plate readings and they look good, almost done, will stop at end of day and compile data into growth curves
SEE: Salt Growth excel
NOTE: normal LB is made with 0.15 M Salt concentrations explaining the optimum grown at the 0.1 M test in salt-free LB
Ex12: Ethyl Acetate Extractions: ELABORATE
Span both of LB and Terrific Broth samples
Stored in -4 C fridge until we have GC time tomorrow night
Ex 10: Talked with Jon and Dan about lack of growth in 5:1 dilution cyano cultures, Probably due to bleaching of bad BG-11. Inoculated new 5:1 culture from joe’s stock and transferred cultures to plate (50uL 7942 and 1uL pmmBRraA) (50uL 7942 control on cm – BG11 plates). Waited 2hours for DNA to absorb then plated and put in light room
If works will give cyanobacteria chloramphenicol resistance
Attempted first natural transformation of PCC 7942 with pMMBRraA (50uL of culture and 1uL of DNA on BG11+CM plates, let sit 2 hours before plating) - NP
- ethyl acetate extraction for GC (3.5 mL induced culture and 1.75mL ethyl acetate, not GC grade, mixed for 5 mins, centrifuged at 2000 rpm for 10 mins then removed 450uL of supernatant ethyl acetate and put into GC vials) - NP
