Team:Reading/Protocols

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<!--Protocols  -->
 
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<tr><td><h3><font color="#558e2b"> A note on protocols </font></h3></td>
 
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<td> <h3 id="intro"><font color="#558e2b"> Contents </font></h3></td>
 
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<!-- Introduction to the Protocols Page -->
 
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A little introduction to our protocols: stuff you need to know, like that we often tweak these protocols in our lab books.
 
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<h1><b><center><font size="6" color="red">DO NOT EDIT THIS PAGE. GO TO THE GOOGLE DOC</font></b></h1>
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<!-- Contents - add stuff here to add to the contents page -->
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<li><a href="#intro">A Note on Protocols</a></li>
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<li><a href="#protocols">The Goods</a></li>
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<ul>
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    <li><a href="#exampleone">Example one</a></li>
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    <li><a href="#exampletwo">Example two</a></li>
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<li><a href="#acknowledge">Acknowledgements</a></li>
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<li><a href="#references">References</a></li>
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<!-- The Protocols -->
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<td colspan="3"><h3 id="protocols"> <font color="#558e2b">The Goods</font> </h3>
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Here are all of the protocols.
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<!-- Miniprep -->
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<p id="exampleone"><font color="#558e2b"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></font></p>
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<ol>
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  <li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
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  <li>Discard supernatant by pipetting off ensuring not to disturb the pellet
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  <li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
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  <li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
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  <li>Centrifuge at 13,000 rpm for 5 minutes
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  <li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
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  <li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
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  <li>Discard the flow through
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  <li>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
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  <li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
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  <li>Incubate at room temperature for 2 minutes
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  <li>Centrifuge for 2 minutes at 13,000 rpm
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<p id="exampletwo"><font color="#558e2b"><i>Making glycerol stock</i></font></p>
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<p>This protocol is adapted from 2 freely available protocols<sup><a href=”#Reference1”>1</a>, <a href=”#Reference2”>2</a></sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet
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  <li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
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  <li>Centrifuge at 13,000 rpm for 2 minutes
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  <li>Discard supernatant
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  <li>Add 0.5 ml of 60% glycerol stock
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    <li>240 ml of glycerol
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    <li>160 ml nano pure water
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    <li>mix together and autoclave
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  <li>freeze at -80℃
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<p id="exampletwo"><font color="#558e2b"><i>Taking optical density of culture</i></font></p>
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<p>Recommendation for taking OD for monitoring growth is either OD<sub>730</sub><sup><a href=”Reference3”>3</a></sup> or OD<sub>750</sub><sup><a href=”#Reference4”>4</a></sup>. Some recommend taking OD<sub>730</sub> at no higher than 0.4 because of problems with light scattering<sup>REFERENCE</sup>. We chose to measure at growth OD<sub>750</sub> to keep in line with other high-profile papers on Synechocystis<sup>4</sup>.
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<li>Set spectrophotometer to measure at OD<sub>750</sub>
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<li>Blank with 1 ml of BG-11 in a cuvette
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<li>Measure 1 ml of culture
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<li>If OD is over 1 dilute the 235 ul of culture in 750 ul of BG-11
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<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 108 cells mL<sup>-1 5</sup>.
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<p id=“PCR”><font color="#558e2b"><i>PCR</i></font></p>
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<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.
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<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.
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<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>REFERENCE</sup>
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<p>PCR program program as follows:
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<ol>
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<li> Start:
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<il>95℃ for 30 seconds
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</ul>
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<li>35 cycles:
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95℃ for 10 seconds
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56℃ for 15 seconds
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72℃ for 70 seconds
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<li>Final
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72℃ for 5 minutes
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68℃ for 10 minutes
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<p>All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup>REFERENCE</sup>
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<p id="exampletwo"><font color="#558e2b"><i>Transformation into E. coli</i></font></p>
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<li>Thaw tubes of competent cells on ice and transfer 50 ul to a pre-chilled 1.5 ml Eppendorf
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<li>Add 5 ul of DNA using a sterile pipette tip
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<li>Flick the tubes to mix and then store on ice for 30 minutes
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<li>Heat shock in a water bath at 42℃ for 1 minute
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<li>Incubate on ice for 5 minutes
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<li>Add 450 ul of SOC (we used LB instead)
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<li>Place tubes horizontally at 37℃ for 2 hours on a shaker at 250 rpm
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<li>Invert Eppendorfs containing the cells several times
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<li>Plate out and incubate overnight at 37℃
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<p> Notes on transformation efficency
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<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). 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>REFERENCE</sup>.
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<p id="exampletwo"><font color="#558e2b"><i>Nanodrop</i></font></p>
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<p>Nanodrop is used to check concentration in DNA often from a miniprep
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<ol>
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<li>Set the Nanodrop to measure DNA
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<li>Blank the Nanodrop by placing 2 ul of PCR water on the stage and pressing blank
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<li>Add 2 ul of sample and measure. Measurement should be in ng/ul
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<p id="exampletwo"><font color="#558e2b"><i>Selection plates</i></font></p>
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<p>BG-11 plates are used to grow Cyanobacteria. Antibiotics are added as needed for selection. 1.5% agar is used.
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<li>Add 7.55 g of agar to 500 ml BG-11 and autoclave to sterilise
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<li>If making kanamycin plates cool to ~55℃ and add 50 ug/ml
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<li>Agar melted in steamer and kept at 55℃ until needed for pouring
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<p> If making a kanamycin cap:
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<li>0.6% agar w/v
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<li>Cool BG-11 to ~55ºC and add 0.5mg/ml kan
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<li>Add ~3ml to a plain BG-11 plate with transformed colonies on it
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<li>Leave for >1 week for Kan selection to occur
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<p id="exampletwo"><font color="#558e2b"><i>Cyanobacteria Transformation</i></font></p>
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<p>Protocol to transform DNA into cyanobacteria
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<li>Make a fresh culture to OD<sub>730</sub>=0.2 to 0.3 and grow for 3 days
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<li>Centrifuge 1.5 ml of culture at 4000g for 10 min. Remove supernatant
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<li>Repeat step 2
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<li>Add 1 ml BG-11, resuspend to wash, centrifuge at 4000g for 10 min, remove supernatant
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<li>Add 200ml fresh BG-11
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<li>4ug plasmid A DNA is added (at a concentration of at least 100ng/ul)
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<li>Incubate for 24 under light on a shaker at ~100 rpm
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<li>Plate the full 200ml and leave for 1-2 days
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<li>Add 3-4ml kan 0.6% agar BG-11
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<li>Leave under light for >1 week
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<td colspan="3"><h3 id="references"><font color="#558e2b">References</font></h3>
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<a href=”Reference1”><p>1. http://www.people.vcu.edu/~pli/Protocols/Plasmid%20Preparation.pdf</a>
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<a href=”Reference2”><p>2. http://openwetware.org/wiki/Making_a_long_term_stock_of_bacteria</a>
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<a href=”Reference3”><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.</a>
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<a href=”Reference4”><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).</a>
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<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.
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<td colspan="3"><h3 id="acknowledge"><font color="#558e2b">Acknowledgements</font></h3>
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Everyone we need to thank for help with protocols.
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