Team:University of Lethbridge/Notebook/Project1September

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Selina, Christa, Munima
Created 10 mL sterile 100 mM theophylline solution. Aliquotted into microfuge tubes and placed in cabinet in teaching lab.

Note: higher solubility in EtOH (see Merck index) but will only reach 100 mM in a super-saturated H20 solution (heat up to 50C directly before use).

Christa, Selina, Munima
Made motility media tubes (~20 blank, ~12 with 1 mM theophylline, ~10 with 0.25 mM theophylline, 1 with 50 mM theophylline). Attempts to make 'layered' motility media stab tubes was a complete disaster.

Labeled tubes and placed in teaching lab beside LB culture tubes - DO NOT MIX THESE UP!!!!

Selina
Counted colonies from previous sucessful competency/transformation attempt (Aug. 25/08).

- CaCl2 treated DH5a + pUC19 = ~3 colonies/uL (~300 colonies in 100 uL) - RP1616 + pUC19 = ~2 colonies/uL (~ 200 colonies in 100 uL)

- CaCl2 treated DH5a + pTopp = ~1.8 colonies/uL (~450 colonies in 250 uL) - RP1616 + pTopp = ~0.2 colonies/uL (~ 20 colonies in 100 uL)

Christa, Munima, John
Objective: Assess motility with our newly made motility media (Sept. 11/08).

Stabbed tubes of 0 mM, 0.25 mM, 1 with 0.5 mM and 1 mM [theophylline] with RP1616 (from -80C glycerol stock) or DH5alpha + pTopp from Aug. 25/08 plate for negative and positive control, respectively.

Incubated at @ 37 C overnight. Motility will be assessed after 48 hours.

Christa
Objective: Glycerol stock cells plated on August 25 & 26, 2008

Subcultured colony from RP 1616 CaCl2 + pTopp from August 26, 2008 plate (single colony) into 1- 5mL culture tube containing 100ug/mL amp. Subcultured colonies from DH5a + pTopp from August 25, 2008 plate into 2- 5mL culture tubes containg 100ug/mL amp. Left in shaker incubator at 37 C overnight.

Munima, Christa, Selina
Objective: Assess the results of motility media stab tubes.

From left to right:

DH5a+pTopp (from Selina's Aug. 25/08 plate) - 0 mM of theophylline - 0.25 mM of theophylline - 1 mM of theophylline RP1616 - 0 mM of theophylline - 0.25 mM of theophylline - 0.50 mM of theophylline - 1 mM of theophylline



Overall results: These positive and negative controls look basically the same. However, the positive controls do not look as they are supposed to. These results are inconclusive. Obtain the controls that the bio labs use and retry the stab tubes.

Christa
Objective: Glycerol stock cells plated on August 25 & 26, 2008

No growth observed from the colony subcultured from RP1616 CaCl2 + pTopp. Left in shaker incubator overnight at 37 C.

Growth observed in culture tubes contain colonies from DH5a + pTopp. Made 4 glycerol stocks of these tubes, stored in the Wieden - 80 C labelled iGEM DH5a + pTopp September 25/08.

Christa
Objective: To transform ligated CheZ into DH5a

Did the transformation and then plated on LB + kan overnight at 37 C

Christa, Munima
Objective: To PCR ligated product from September 27th

Ran a colony PCR on DH5a cells with ligated CheZ. Ran 5 reactions: Colony 1, Colony 2, Colony 3, Negative (water), Positive (pTopp) - Tempate DNA 1uL - 10X Econo Taq Buffer 2.5uL - dNTP mix 1uL - Primer 1 5uL - Primer 2 5uL - Econo Taq 0.25uL - ddH2O 19.75uL

Cycling Conditions: - Incubate PCR Reactions 2 min at 94 C - Denature 30 sec at 94 C  - Anneal 30 sec at 47 C  - Extend 1 min/kb at 72 C  - Final extension 7 min at 72 C  - Hold indefinitely at 4 C

Lids popped off PCR tubes; we will retry later

Inoculated LB + 50ug/mL Kam liquid media with DH5a with CheZ in pzero

Munima
Objective: To run colony PCR on ligated CheZ in DH5a

Plasmid prepped pZero and CheZ plasmmids form DH5a that Christa and Nathan transformed on September 27 from subculture that Munima and Christa inoculated on September 29. One plasmid prep done (1.5mL) culture of each of the three colonies using Qiaqen Mini Prep Kit. Plasmid prep tubes (50uL) are in iGEM -20 C freezer and labelled DH5a, pZero, CheZ Sept 30/08.