Team:Reading/Protocols
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+ | <!--Protocols --> | ||
+ | <tr> | ||
+ | <td><h3 class="title">A note on protocols</h3></td> | ||
+ | <td width="20%"></td> | ||
+ | <td> <h3 class="title">Contents</h3></td> | ||
+ | </tr> | ||
+ | |||
+ | <!-- Introduction to the Protocols Page --> | ||
+ | <tr> | ||
+ | <td width="50%" 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></td> | ||
+ | <td></td> | ||
+ | |||
+ | <!-- Contents --> | ||
+ | <td width="27%" valign="top"> | ||
+ | <ol> | ||
+ | <li><a href="#">A Note on Protocols</a> | ||
+ | <li><a href="#protocols">Protocols</a> | ||
+ | <ul> | ||
+ | <li><a href="#prot1">Miniprep</a> | ||
+ | <li><a href="#prot2">Glycerol stock</a> | ||
+ | <li><a href="#prot3">Optical Density</a> | ||
+ | <li><a href="#prot4">PCR</a> | ||
+ | <li id=protocols><a href="#prot5">Transformation (E. coli)</a> | ||
+ | <li><a href="#prot6">Nanodrop</a> | ||
+ | <li id=prot1><a href="#prot7">BG-11 plates</a> | ||
+ | <li><a href="#prot8">Cyanobacteria Transformation</a> | ||
+ | <li><a href="#prot9">Biobrick Assembly</a></ul> | ||
+ | </ul> | ||
+ | <li><a href="#references">References</a> | ||
+ | <li><a href="#acknowledge">Acknowledgements</a> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <!-- Spacer line --> | ||
+ | <tr><td colspan="3" height="15px"></td></tr> | ||
+ | <tr><td bgColor="#CCCCCC" colspan="3" height="1px"></tr> | ||
+ | <tr><td colspan="3" height="5px"></td></tr> | ||
+ | |||
+ | <!-- The Protocols --> | ||
+ | <tr> | ||
+ | <td colspan="3"> | ||
+ | <h3 class="title">Protocols</h3> | ||
+ | |||
+ | <!--Miniprep --> | ||
+ | <br> | ||
+ | <p class="title"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></p> | ||
+ | <ol> | ||
+ | <li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes | ||
+ | <li>Discard supernatant by pipetting off ensuring not to disturb the pellet | ||
+ | <li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes | ||
+ | <li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times | ||
+ | <li>Centrifuge at 13,000 rpm for 5 minutes | ||
+ | <li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate | ||
+ | <li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm | ||
+ | <li>Discard the flow through | ||
+ | <li>Add 500 µl of wash solution to the column | ||
+ | <li>Centrifuge for 1 minute at 13,000 rpm | ||
+ | <li id=prot2>Discard flow through | ||
+ | <li>Add 500 µl of wash solution to the column | ||
+ | <li>Centrifuge for 1 minute at 13,000 rpm | ||
+ | <li>Discard flow through | ||
+ | <li>Centrifuge for 1 minute at 13,000 rpm | ||
+ | <li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube | ||
+ | <li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette | ||
+ | <li>Incubate at room temperature for 2 minutes | ||
+ | <li>Centrifuge for 2 minutes at 13,000 rpm | ||
+ | </ol> | ||
+ | |||
+ | <!-- Glycerol stock --> | ||
+ | <br> | ||
+ | <p class="title"><i>Making glycerol stock</i></p> | ||
+ | <p id=prot3>This protocol is adapted from 2 freely available protocols<sup><a href="#references">1</a>, <a href="#references">2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet</p> | ||
+ | <ol> | ||
+ | <li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips | ||
+ | <li>Centrifuge at 13,000 rpm for 2 minutes | ||
+ | <li>Discard supernatant | ||
+ | <li>Add 0.5 ml of 60% glycerol stock | ||
+ | <ol style="list-style: lower-roman outside"> | ||
+ | <li>240 ml of glycerol | ||
+ | <li>160 ml nano pure water | ||
+ | <li>mix together and autoclave | ||
+ | </ol> | ||
+ | <li>freeze at -80℃ | ||
+ | </ol> | ||
+ | |||
+ | <!-- Optical Density --> | ||
+ | <br> | ||
+ | <p class="title"><i>Taking optical density of culture</i></p> | ||
+ | <p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href="#references">3</a></sup> or OD<sub>750</sub><sup><a href="#references">4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup id=prot4><a href=”#references”>4</a></sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup><a href="#references">4</a></sup>.</p> | ||
+ | <ol> | ||
+ | <li>Set spectrophotometer to measure at OD<sub>750</sub> | ||
+ | <li>Blank with 1 ml of BG-11 in a cuvette | ||
+ | <li>Measure 1 ml of culture | ||
+ | <li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11 | ||
+ | </ol> | ||
+ | <p>Converting OD<sub>750</sub> 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 10<sup>8</sup> cells mL<sup><a href="#references">5</a></sup>.</p> | ||
+ | |||
+ | <!-- PCR --> | ||
+ | <br> | ||
+ | <p class="title"><i>PCR</i></p> | ||
+ | <p>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.</p> | ||
+ | <p> 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. </p> | ||
+ | <p>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 <sup><a href="#references">6</a></sup></p> | ||
+ | <p>PCR program program as follows:</p> | ||
+ | <ol> | ||
+ | <li> Start: | ||
+ | <ul> | ||
+ | <li>95℃ for 30 seconds | ||
+ | </ul> | ||
+ | <li id=prot5>35 cycles: | ||
+ | <ul> | ||
+ | <li>95℃ for 10 seconds | ||
+ | <li>56℃ for 15 seconds | ||
+ | <li>72℃ for 70 seconds | ||
+ | </ul> | ||
+ | <li>Final | ||
+ | <ul> | ||
+ | <li>72℃ for 5 minutes | ||
+ | <li>68℃ for 10 minutes | ||
+ | </ul> | ||
+ | </ol> | ||
+ | <p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup><a href="#references">7</a></sup></p> | ||
+ | |||
+ | <!-- Transformation (E. coli) --> | ||
+ | <br> | ||
+ | <p class="title"><i>Transformation into E. coli</i></p> | ||
+ | <p> | ||
+ | <ol> | ||
+ | <li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf | ||
+ | <li>Add 5 ul of DNA using a sterile pipette tip | ||
+ | <li>Flick the tubes to mix and then store on ice for 30 minutes | ||
+ | <li>Heat shock in a water bath at 42℃ for 1 minute | ||
+ | <li>Incubate on ice for 5 minutes | ||
+ | <li id=prot6>Add 450 ul of SOC (we used LB instead) | ||
+ | <li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm | ||
+ | <li>Invert Eppendorfs containing the cells several times | ||
+ | <li>Plate out and incubate overnight at 37℃ | ||
+ | </ol> | ||
+ | <p> Notes on transformation efficency</p> | ||
+ | <p>Expected transformation efficiency is 1 x 10<sup>6</sup> 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). <a id=prot7 />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 <sup><a href="#references">8</a></sup>.</p> | ||
+ | |||
+ | <!-- Nanodrop --> | ||
+ | <br> | ||
+ | <p class="title"><i>Nanodrop</i></p> | ||
+ | <p>Nanodrop is used to check concentration in DNA often from a miniprep</p> | ||
+ | <ol> | ||
+ | <li>Set the Nanodrop to measure DNA | ||
+ | <li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank | ||
+ | <li>Add 2 ul of sample and measure. Measurement should be in ng/ul | ||
+ | </ol> | ||
+ | |||
+ | <!-- BG-11 plates --> | ||
+ | <br> | ||
+ | <p class="title"><i>BG-11 plates</i></p> | ||
+ | <p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.</p> | ||
+ | <ol> | ||
+ | <li id=prot8>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise | ||
+ | <li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml | ||
+ | <li>Agar melted in steamer and kept at 55℃ until needed for pouring | ||
+ | </ol> | ||
+ | <p> If making a kanamycin cap:</p> | ||
+ | <ol> | ||
+ | <li>0.6% agar w/v | ||
+ | <li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan | ||
+ | <li>Add ~3ml to a plain BG-11 plate with transformed colonies on it | ||
+ | <li>Leave for >1 week for Kan selection to occur | ||
+ | </ol> | ||
+ | |||
+ | <!-- Cyanobacteria Transformation --> | ||
+ | <br> | ||
+ | <p class="title"><i>Cyanobacteria Transformation</i></p> | ||
+ | <p>Protocol to transform DNA into cyanobacteria</p> | ||
+ | <ol> | ||
+ | <li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days | ||
+ | <li id=prot9>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant | ||
+ | <li>Repeat step 2 | ||
+ | <li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant | ||
+ | <li>Add 200ml fresh BG-11 | ||
+ | <li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul) | ||
+ | <li>Incubate for 24 under light on a shaker at ~100 rpm | ||
+ | <li>Plate the full 200ml and leave for 1-2 days | ||
+ | <li>Add 3-4ml kan 0.6% agar BG-11 | ||
+ | <li>Leave under light for >1 week | ||
+ | </ol> | ||
+ | |||
+ | <!-- Biobrick Assembly --> | ||
+ | <br> | ||
+ | <p class="title"><i>Biobrick Assembly</i></p> | ||
+ | <p>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:</p> | ||
+ | <ol> | ||
+ | <li>In a PCR tube add 20 ul of water and either 5 ul of your construct or 2 ul of plasmid backbone | ||
+ | <li>To the same tube add 2.5 ul of NED buffer | ||
+ | <li>Add appropriate restriction enzymes. In our case 1 ul of EcoR1 and 1 ul of Pst1 | ||
+ | <li>Incubate the tubes at 37℃ for 15 minutes | ||
+ | <li>Then incubate at 80℃ for 20 minutes | ||
+ | <li id=references>Add 5 ul of water to a new tube followed by 2 ul of the digested construct and backbone | ||
+ | <li>Add 2 ul of 10X T4 DNA ligase restriction buffer | ||
+ | <li>Add 1 ul of T4 DNA ligase | ||
+ | <li>Incubate at room temperature for 10 minutes | ||
+ | <li>Incubate reaction mixture at 80℃ for 20 minutes. This step inactivates the enzyme | ||
+ | </ol> | ||
+ | <p>This DNA can now be used for transformation</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <!-- Spacer line --> | ||
+ | <tr> <td colspan="3" height="15px"> </td></tr> | ||
+ | <tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr> | ||
+ | <tr> <td colspan="3" height="5px"> </td></tr> | ||
+ | |||
+ | <!-- References Section --> | ||
+ | <tr> | ||
+ | <td colspan="3"><h3 class="title">References</h3> | ||
+ | <p>1. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</p> | ||
+ | <p>2. http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</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>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>http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf</p> | ||
+ | <p>https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <!-- Spacer line --> | ||
+ | <tr> <td colspan="3" height="15px"> </td></tr> | ||
+ | <tr><td bgColor="#CCCCCC" colspan="3" height="1px"> </tr> | ||
+ | <tr> <td colspan="3" height="5px"> </td></tr> | ||
+ | |||
+ | <!-- Acknowledgements section --> | ||
+ | <tr> | ||
+ | <td colspan="3"><h3 class="title">Acknowledgements</h3> | ||
+ | <p>Everyone we need to thank for help with protocols.</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | </html> | ||
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Revision as of 20:16, 16 October 2014
University of Reading | |||||||||||
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A note on protocols |
Contents |
|
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. |
||
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
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. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf 2. 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. http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987 |
||
AcknowledgementsEveryone we need to thank for help with protocols. |
rusynbioigem@gmail.com |