Team:EPF-Lausanne/Protocols

From 2011.igem.org

(Difference between revisions)
(→Cloning, assembly, and mutations)
 
(50 intermediate revisions not shown)
Line 1: Line 1:
-
== PDMS two layer device fabrication ==
+
{{:Team:EPF-Lausanne/Templates/Header|title=Protocols}}
-
made on 16.05.2011
+
 +
== Molecular Biology ==
-
<p>'''''Materials: </p>
+
=== Products and stock preparation ===
-
----
+
* [[Team:EPF-Lausanne/Protocols/Primers preparation|Primer preparation]]: initial dilution of ordered primers.
-
<p>    </p>
+
* [[Team:EPF-Lausanne/Protocols/Competent cells|Competent Cells]]: prepare cells for plasmid uptake.
-
<p>• flow and control layer molds</p>
+
* [[Team:EPF-Lausanne/Protocols/Competent cells. Protocol II|Competent Cells. Protocol II (Inoue method)]].
-
<p>• Sylgard 184 silicone elastomer kit: Base part and a Curing agent</p>
+
* [[Team:EPF-Lausanne/Protocols/Glycerol stock|Glycerol stock]]: stock transformed cells at -80°C.
-
<p>• TMCS</p>
+
* [[Team:EPF-Lausanne/Protocols/Agar Plates|Agar plate preparation]]: preparing agar plates for cell cultures.
 +
* [[Team:EPF-Lausanne/Protocols/Autoclave|Autoclave]]: to sterilize solutions and glassware.
-
<br>
+
=== Cloning, assembly, and mutations ===
-
<p>'''''Method:</p>
+
* [[Team:EPF-Lausanne/Protocols/Transformation|Transformation]]: introduce foreign plasmid into competent cells.
 +
* [[Team:EPF-Lausanne/Protocols/Plating|Plating]]: plate transformed cells
 +
* [[Team:EPF-Lausanne/Protocols/Liquid cultures|Liquid cultures]]: when cells have grown on plates, put them in liquid cultures
 +
* [[Team:EPF-Lausanne/Protocols/Gibson assembly|Gibson assembly]].
 +
* [[Team:EPF-Lausanne/Protocols/TetR|Linear template- TetR]].
 +
* [[Team:EPF-Lausanne/Protocols/TetR Extension PCR|tetR Overlap Extension PCR]]: induce specific mutations on tetR linear template.
 +
* [[Team:EPF-Lausanne/Protocols/Site-specific mutagenesis|tetR Site-specific mutagenesis]]: induce site-specific mutations on a plasmid containing tetR.
 +
* [[Team:EPF-Lausanne/Protocols/Gel_Electrophoresis|Agarose gel electrophoresis]]: DNA samples analysis (can be followed by band purification).
 +
* [[Team:EPF-Lausanne/Protocols/Colony PCR|Colony PCR]]: Analyze the plated transformed cells, to see if Gibson assembly worked
 +
* [[Team:EPF-Lausanne/Protocols/T7-ext|T7 extension PCR]]: Put T7 promoter upstream of a gene
 +
* [[Team:EPF-Lausanne/Protocols/bluntends|Blunt-End Cloning]]: Making Blunt Ends
-
<p>  </p>
+
=== DNA recovery ===
-
----
+
-
<p>• Place molds into a TMCS vapor chamber</p>
+
* [[Team:EPF-Lausanne/Protocols/Miniprep|Plasmid preparation - Miniprep]]: recover plasmids from a bacterial culture.
-
<p>• Control layer mixture: 20g Base + 4g Curing agent</p>
+
* [[Team:EPF-Lausanne/Protocols/Gel purification|Gel purification]]: recover DNA separated by electrophoresis
-
<p>• Mix for 1 minute, degas for 2 minutes (standard protocol)</p>
+
-
<p>• pour onto control layer mold and place mold in vacuum chamber</p>
+
-
<p>• Flow layer mixture: 20g Base + 1g Curing agent</p>
+
-
<p>• Mix for 1 minute and degas for 2 minutes (standard protocol)</p>
+
-
<p>• Spin coat onto flow layer at 2200-2400rpm for 35secs (so far you can use my protocol on a spin coater)</p>
+
-
<p>• Remove control layer mold from vacuum chamber, making sure no bubbles are left on the surface (remove with a toothpick if you see some )</p>
+
-
<p>• Place the control and flow layer in a 80C convection oven and incubate for 30 minutes (timing is critical here!)</p>
+
-
<p>• Remove casts from oven, cut out control layer, punch holes, and align to flow layer</p>
+
-
<p>• Put aligned device back into 80C oven and incubate for at least 90 minutes (and here you can increase the backing time)</p>
+
-
<p>• Remove devices from oven and punch flow layer holes</p>
+
-
</div>
+
== Biochemistry ==
 +
* [[Team:EPF-Lausanne/Protocols/IPTG test|IPTG test]]: test expression of a gene downstream from Plac
 +
== Microfluidics ==
-
== MITOMI: Protein – DNA interactions ==
+
* [[Team:EPF-Lausanne/Protocols/PDMS_two_layer_device_fabrication|PDMS two layer device fabrication]]
-
made on 16.05.2011
+
* [[Team:EPF-Lausanne/Protocols/Master microfabrication for PDMS replica molding |Master microfabrication for PDMS replica molding]]
 +
* [[Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA|MITOMI: Protein – DNA interactions]]
 +
* [[Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis|Klenow dsDNA synthesis]]
 +
== Worm culture ==
 +
* [[Team:EPF-Lausanne/Protocols/MG_Agar_Plates|Agar plate preparation]]: preparing MG Agar plates for worm cultures.
-
<p>'''''Purpose: </p>
+
{{:Team:EPF-Lausanne/Templates/Footer}}
-
----
+
-
<p>  To detect potential protein-DNA interactions </p>
+
-
<p>''''' Chip:</p>
+
-
----
+
-
 
+
-
MITOMI device bonded to epoxy slide spotted with DNA samples, bonded at 40C overnight (alternatively @room temperature)
+
-
+
-
<br>
+
-
<p>''''' Method:</p>
+
-
<p>• Load all control lines with dH2O, at ~5 psi, check that all valves work</p>
+
-
<p> '' Usually “B” valves are starting to load earlier (leave the button unpressurised prior to the pressure increase, it tends to stick to the slide.)</p>
+
-
<p>  Increase the pressure in both “A” and “B” valves till ~13-15 psi and make sure that you can see that all chambers are separated. If no – increase the pressure''</p>
+
-
<p>• 2mg/ml BSA-bio @ 5psi for 20 minutes; chambers (B3): closed</p>
+
-
<p>• '' You can prepare the ITT mix meanwhile and put it at 25C for 3-4 hours (it takes that long to express your TF with a GFP tag)'' </p>
+
-
<p>• PBS for ~2 minutes (or as long as the preparation for next step takes)</p>
+
-
<p>• 500ug/ml NA PBS for 20 minutes</p>
+
-
<p>• PBS for ~5 minutes</p>
+
-
<p>• Close button, continue PBS for 1-2minutes making sure button is closedv
+
-
<p>• 2mg/ml BSA biotin for 20 minutes</p>
+
-
<p>• PBS for 10 minutes</p>
+
-
<p>• Antibody-GFP-biotin in PBS for 4-5 minutes</p>
+
-
<p>• open button, continue antibody for 20 minutes</p>
+
-
<p>• PBS for ~10 minutes</p>
+
-
<p>• flow the expression mix with expressed protein for 10-15 minutes, when  ~2-3 cm of mix left in the inlet tube :</p>
+
-
<p>• Close outlet B4 and open B3 (isolates spotted DNA in a chamber) and load chambers with the same expression mix</p>
+
-
<p>            ''To do so you should close the outlet first (“B4”) and continue flow. Stop the flow as soon as all your chambers with DNA will be filled. Do not wait for too long or DNA will diffuse to the neighbor chamber''</p>
+
-
<p>• close chambers</p>
+
-
<p>• close sandwich</p>
+
-
<p>• open chamber</p>
+
-
<p>• incubate TF with target DNA at RT for 30-60 minutes (to reach a thermodynamic equilibrium)</p>
+
-
<p>• scan using Arrayworx</p>
+
-
<p>• close button</p>
+
-
<p>• close chamber</p>
+
-
<p>• open sandwich</p>
+
-
<p>• PBS for 10-15 minutes</p>
+
-
<p>• scan using Arrayworx to detect the interacting sequences</p>
+

Latest revision as of 15:35, 16 October 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Protocols"