Team:Reading/Protocols
From 2014.igem.org
(18 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
{{Head}} | {{Head}} | ||
<html> | <html> | ||
- | <br><br><br><br><br><br><br><br><br> | + | <br><br><br><br><br><br><br><br><br><br> |
- | <table | + | <table> |
<!--Protocols --> | <!--Protocols --> | ||
<tr> | <tr> | ||
<td><h3 class="title">A note on protocols</h3></td> | <td><h3 class="title">A note on protocols</h3></td> | ||
- | |||
- | |||
</tr> | </tr> | ||
<!-- Introduction to the Protocols Page --> | <!-- Introduction to the Protocols Page --> | ||
<tr> | <tr> | ||
- | <td width=" | + | <td width="80%" valign="top"> <p>Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project.</p> |
- | <br></ | + | <br> |
- | < | + | <p align="center"><img src="https://static.igem.org/mediawiki/2014/0/0d/Oscar_doing_stuff.jpg" width="700"/></p> |
+ | </td> | ||
<!-- Contents --> | <!-- Contents --> | ||
<td width="27%" valign="top"> | <td width="27%" valign="top"> | ||
+ | <h3 class="title">Contents</h3> | ||
<ol> | <ol> | ||
<li><a href="#">A Note on Protocols</a> | <li><a href="#">A Note on Protocols</a> | ||
Line 34: | Line 34: | ||
</ul> | </ul> | ||
<li><a href="#references">References</a> | <li><a href="#references">References</a> | ||
- | |||
</ol> | </ol> | ||
</td> | </td> | ||
Line 180: | Line 179: | ||
<ol> | <ol> | ||
<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days | <li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days | ||
- | <li | + | <li>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant |
<li>Repeat step 2 | <li>Repeat step 2 | ||
<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant | <li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant | ||
Line 190: | Line 189: | ||
<li>Leave under light for >1 week | <li>Leave under light for >1 week | ||
</ol> | </ol> | ||
+ | <p id=prot9> After many attempts to transform with Cyanobacteria we refined this protocol | ||
+ | <ol> | ||
+ | <li>Grow 6803 to ~0.435 | ||
+ | <li>Take 1 ml, re-wash with BG-11 | ||
+ | <li>Re-suspend in 90ml BG-11 | ||
+ | <li>Plasmid DNA at ~270ng/ul | ||
+ | <li>add 8ul to each tube | ||
+ | <li>place in 34ºC water bath, unshaken, in the dark, for 3 hours and 20 minutes | ||
+ | <li>transfer to warm room at 28ºC and leave overnight | ||
+ | <li>plate on Kan50 and leave under light, agar-side down | ||
+ | </ol> | ||
<!-- Biobrick Assembly --> | <!-- Biobrick Assembly --> | ||
Line 219: | Line 229: | ||
<tr> | <tr> | ||
<td colspan="3"><h3 class="title">References</h3> | <td colspan="3"><h3 class="title">References</h3> | ||
- | <p>1. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</p> | + | <p>1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: <a href="http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf" title="To make Glycerol Stocks of Plasmids">http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a> </p> |
- | <p>2. http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</p> | + | <p>2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: <a href="http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria" title="Making a long term stock of bacteria">http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a> </p> |
<p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p> | <p>3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At <http://link.springer.com/protocol/10.1007/978-1-60761-925-3_22>. Accessed 20/08/2014.</p> | ||
<p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p> | <p>4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).</p> | ||
- | <p>5 | + | <p>5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p> |
<p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p> | <p>6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999.</p> | ||
- | <p>http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</p> | + | <p>7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: <a href="http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf" title="ThermoScientific GeneJET PCR Purification Kit #K0701, #K0702">http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</a></p> |
- | <p>https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</p> | + | <p>8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: <a href="https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987" title="High Efficiency Transformation Protocol (C2987H/C2987I)">https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</a></p> |
</td> | </td> | ||
</tr> | </tr> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
</table> | </table> | ||
</html> | </html> | ||
{{Tail}} | {{Tail}} |
Latest revision as of 01:02, 18 October 2014
University of Reading | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
|
A note on protocols |
||
Here we present a selection of the most important protocols we gathered over the course of our lab work. Many are adapted from freely available protocols and in these case a link is provided to the original. Any modifications we made are through trial and error experience on our part and may therefore not translate to your project. Acknowledgments are also listed thanking people who helped us with our project. |
Contents |
|
ProtocolsIsolation of plasmid DNA from bacteria (miniprep)
Making glycerol stock This protocol is adapted from 2 freely available protocols1, 2. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet
Taking optical density of culture Recommendation for taking OD for monitoring growth is either OD7303 or OD7504. Some recommend taking OD730 at no higher than 0.4 because of problems with light scattering4. We chose to measure at growth OD750 to keep in line with other high-profile papers on Synechocystis4.
Converting OD750 to cell density For conversion of cell densities to numbers of cells, we have used the relationship OD750 = 1 (a.u) corresponding to 1.6 x 108 cells mL5. PCR PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid. Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage. 2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix 6 PCR program program as follows:
All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol7 Transformation into E. coli
Notes on transformation efficency Expected transformation efficiency is 1 x 106 cfu/ug of pUC19 DNA, but we should expect a 2-fold decrease in efficiency due to use of LB instead of SOC (a derivative of super optimal broth, SOB). We should also expect a decrease because we thawed the frozen competent cells at a temperature above 0ºC. Ideally they should be thawed on ice, or by hand if needed 8. Nanodrop Nanodrop is used to check concentration in DNA often from a miniprep
BG-11 plates BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.
If making a kanamycin cap:
Cyanobacteria Transformation Protocol to transform DNA into cyanobacteria
After many attempts to transform with Cyanobacteria we refined this protocol
Biobrick Assembly Protocol for inserting a construct into a plasmid backbone. In the case of BioBrick this will commonly be pSB1C3. Biobrick has 2 stages, a digestion stage and a ligation stage:
This DNA can now be used for transformation |
||
References1. Virginia Commonwealth University. To make Glycerol Stocks of Plasmids. [Online] Available at: http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf 2. OpenWetWare. 2012. Making a long term stock of bacteria. [Online] Available at: http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria 3. Eaton-Rye, J. J. in Photosynth. Res. Protoc. 295–312 (Humana Press, 2011). At 4. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013). 5. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999. 6. Pojidaeva E, Zichenko V, Shestakov SV, Sokolenko A (2004) Involvement of the SppA1 peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol 186: 3991–3999. 7. ThermoScientific. 2013. Thermo Scientific GeneJET PCR Purification Kit #K0701, #K0702. [Online] Available at: http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf 8. New England BioLabs. 2014. High Efficiency Transformation Protocol (C2987H/C2987I). [Online] Available at: https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987 |
rusynbioigem@gmail.com |