Team:Warsaw/Calendar-Main/29 July 2008

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Revision as of 21:11, 11 October 2008


	Attribution analyzer / Interactive list of topics

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains).

Checking if OmpA_omega_A_alpha gives ampicillin resistance
Piotr

Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Measurement of bacterial culture growth (OD) in the evening:

ampicillin concentration (μg/mL):	IPTG concentration (mmol/mL):
ampicillin concentration (μg/mL):	0	0.1	0.25	0.5	0.75	1
25	1.558	1.469	1.587	1.49	1.566	1.311
50	1.425	1.435	1.524	1.055	0.920	0.935
75	1.09	0.989	1.447	0.971	0.951	0.992
100	0.09	0.685	1.378	1.078	0.977	0.992

Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)

Checking OmpA_omega_A_alpha and OmpA_A_alpha expression
Piotr

Spinning
Suspending
Adding of lysis buffer
Boiling
Putting into poliacrylamide gel
Transfer onto nitrocellulose
Blocking
Anti-A antibody binding
Washing
Anti-rabbit antibody binding
Developing with BCIP and NBT

[a photo of the gel is top be put here]

Michał L., Ewa, Marcin

Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.

@@ Line 9: / Line 9: @@
-<h3>Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha</h3>
-<ol><li>DNA fragment of omega_A isolated from gel on 20 July <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digestion</a> with SacI and NotI.</li>
-<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>clean-up</a>Clean-up of digested DNA fragment.</li>
-<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digestion</a> (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha. </li>
-<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp band. </li>
-<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments from 2. and 4. (1 hr)</li>
-<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
-<li>Transformants plating on LB + kanamycin.</li></ol>
-</p>
 <h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br>