Team:Kyoto/Protocol

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{{Kyoto_Foreground}}
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{{Kyoto_Foreground|active_page=method}}
{{Kyoto_Background}}
{{Kyoto_Background}}
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<!-- main -->
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='''Protocol'''=
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<div id="main">
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Listed below our protocol for this project.<br>
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We hope you find them useful!!
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= '''Protocol''' =
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<html><a name="cloning"></a></html>
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=== Medium for drosophila ===
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[[File:Cloning.PNG|link=https://2011.igem.org/Team:Kyoto/Cloning|Cloning]]<br>
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::{| class="wikitable"
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[[File:Medium.PNG|link=https://2011.igem.org/Team:Kyoto/Medium|Medium]]<br>
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|+ [http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]
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[[File:Measurement.PNG|link=https://2011.igem.org/Team:Kyoto/Measurement|Measurement]]
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! width="300" |Materials
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! width="300" |Methods
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|-
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* water : 500 mL
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* dry yeast : 20 g
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* corn flour : 45 g
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* glucose : 50 g
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* agarose : 3.5~5 g
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* propionic acid : 1.5 mL
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* 10 % p-hydroxybenzoate in 70 % Eternol : 5 g
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# Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it.
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# Stir corn flour and glucose with the remaining water.
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# Stir 1 and 2, then autoclave it again.
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# after autoclave, add propionic acid and 10 % p-hydroxybenzoate in 70 % Eternol into it.■
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|}
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<br/>
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===M9 medium===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* water : 1 L
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* Na2HPO4: 6 g
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* KH2PO4 : 3 g
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* NaCl : 0.5 g
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* NH4Cl : 1 g
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* 100 mM MgSO4 : 10 ml
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* 20 % glucose : 10 mL
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* 10 mM CaCl2 : 10 ml
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* 100 mM thiamine-HCl : 10 ml
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* 20 % casamino acid : 10 ml
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# Stir Na2HPO4, KH2PO4, NaCl and NH4Cl with water.
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# After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, 100 mM thiamine-HCl.
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# If you need, add 10 ml filter sterilized 20 % casamino acid.
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|}
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<br/>
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===LB medium===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* water : 1 L
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* Tryptone: 10 g
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* Yeast extract : 5 g
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* NaCl : 10 g
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* Agar : 15g
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# Stir Tryptone, Yeast extract and NaCl with water.
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# If you make LB plates, add agar.
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# Autoclave.
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|}
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<br/>
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===SOB medium===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* water : 100 mL
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* Tryptone: 2 g
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* Yeast extract : 0.5 g
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* 5 M NaCl : 200 ul
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* 3 M KCl : 84 ul
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* 1 M MgSO4 : 1 ml
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* 1 M MgCl2 : 1 ml
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# Stir Tryptone and Yeast extract with water.
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# Add 200 ul 5 M NaCl and 84 ul 3 M KCl.
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# After autoclave, add 1 ml filter sterilized 10 mM MgSO4 and 10 mM MgCl2.
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|}
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<br/>
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===SOC medium===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* SOB : 100 ml
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* 2 M glucose: 1 ml
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# Add 2 M glucose to 100 ml SOB.
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|}
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<br/>
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===Buffer TB===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* water : 200 ml
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* PIPES : 3 g
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* CaCl2・2H2O : 0.22 g
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* 3 M KCl : 8.315 ml
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* KOH
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* MnCl2・4H2O : 1.09 g
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# Stir PIPES and CaCl2・2H2O with 100 ml water.
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# Add 8.315 ml 3 M KCl.
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# Add KOH and adjust pH 6.8.
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# Add MnCl2・4H2O.
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# Add water up to 200 ml.
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# Filter sterilize
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|}
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===DNS reaegnt===
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::{| class="wikitable"
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! width="300" |Materials
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! width="300" |Methods
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|-
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* 4.5% NaOH : 30 ml
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* 1% DNS : 88 ml
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* Rochelle salt : 25.5 g
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* 10% NaOH : 2.2 ml
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* Phenol : 1 g
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* Water : 7.8 ml
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* NaHCO3 : 0.69 g
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# Add 1% DNS 88ml and Rochelle salt 25.5 g to 4.5% NaOH 30 ml (A solution)
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# Add Phenol 1 g and water 7.8 ml to 10% NaOH 2.2 ml (B solution)
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# Add NaHCO3 6.9g to B solution 6.9 ml
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# Add A solution 118ml to B solution 6.9 ml
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# Leave 2 days
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# Store in brown bottle
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|}
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<a name="PCR"></a><h4> <span class="mw-headline">PCR</span></h4>
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<a name="Standard_PCR"></a><h5> <span class="mw-headline">Standard PCR</span></h5>
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<ol><li> Dilute template DNA. If the concentration of DNA is 2-100ng/&micro;L, transfer 1&micro;L to a clean tube and add 99&micro;L MilliQ.
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</li><li> Dilute Primer. If the concentration of Primer is X&micro;M, dilute primer X timers and transfer 1&micro;L to a clean tube and add 99&micro;L MilliQ.
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</li><li> Mix the following.
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<ol><li> For use of KOD plus ver2:
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<dl><dt> 25mM MgSO4</dt><dd> 3&micro;L
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</dd><dt> 2mM dNTPs</dt><dd> 5&micro;L
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</dd><dt> 10xBuffer for KOD plus ver.2</dt><dd> 5&micro;L
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</dd><dt> Template DNA  (5ng/&micro;L)</dt><dd> 5&micro;L
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</dd><dt> Primer Forward (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> Primer Reverse (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> KOD plus ver.2</dt><dd> 1&micro;L
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</dd><dt> MilliQ</dt><dd> 28&micro;L
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</dd><dt> Total</dt><dd> 50&micro;L
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</dd></dl>
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</li><li> For use of KOD FX:
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<dl><dt> 2mM dNTPs</dt><dd> 10&micro;L
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</dd><dt> 2xBuffer for KOD FX</dt><dd> 25&micro;L
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</dd><dt> Template DNA</dt><dd> 5&micro;L
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</dd><dt> Primer Forward (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> Primer Reverse (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> KOD FX</dt><dd> 1&micro;L
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</dd><dt> MilliQ</dt><dd> 6&micro;L
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</dd><dt> Total</dt><dd> 50&micro;L
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</dd></dl>
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</li></ol>
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</li><li> (If amplification is not succeeded, try at 4.5 or 6.0&micro;L 25mM MgSO4.)
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</li><li> Let stand for 2min at 94&#x2103;.
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</li><li> 25-40 cycles for 10s at 98&#x2103;, for 30s at Tm-5&#x2103;, and for 1min (1min for 1kb) at 68&#x2103; (Tm is temparature at which primer will dissolve).
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</li><li> At 15&#x2103; forever.
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</li><li> Agarose Gel Electrophoresis for confirmation.
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</li></ol>
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<a name="Screening_PCR"></a><h5> <span class="mw-headline">Screening PCR</span></h5>
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<ol><li> Mix the following (Do on PCR Bench).
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<dl><dt> 10x PCR buffer (TAKARA)</dt><dd> 40&micro;L
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</dd><dt> 2.5mM dNTP</dt><dd> 8&micro;L
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</dd><dt> Primer-1 (10pmol/&micro;L)</dt><dd> 8&micro;L
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</dd><dt> Primer-2 (10pmol/&micro;L)</dt><dd> 8&micro;L
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</dd><dt> Ex Taq HS (TAKARA)</dt><dd> 1.6&micro;L
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</dd><dt> MilliQ&nbsp;</dt><dd> 334&micro;L (to total 400&micro;L)
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</dd></dl>
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</li><li> Dispense 25&micro;L to 15 tubes.
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</li><li> Pick a single colony and transfer it to each tubes.
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</li><li> Suspend the colony.
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</li><li> Let stand for 10min at 90&#x2103;.
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</li><li> 35 cycles for 30s at 94&#x2103;, for 30s 55&#x2103;, and for 1min at 72&#x2103;.
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</li><li> Let stand for 4min at 72&#x2103;.
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</li><li> Add 5mL Loading Buffer to the tubes.
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</li><li> Agalose Gel Electrophoresis for confirmation.
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</li><li> Negative Control: Use nothing.
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</li><li> Positive Control: Use a colony that will yield a product with this primers.
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</li></ol>
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<a name="Mutagenesis_.28Point_mutation.2C_Deletion.2C_Insertion.29"></a><h5> <span class="mw-headline">Mutagenesis (Point mutation, Deletion, Insertion)</span></h5>
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<ol><li> Mix the following.
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<dl><dt> 10xBuffer</dt><dd> 5&micro;L
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</dd><dt> 2mM dNTP</dt><dd> 5&micro;L
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</dd><dt> Primer Forward (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> Primer Reverse (10&micro;M)</dt><dd> 1.5&micro;L
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</dd><dt> Template Plasmid (50ng/&micro;L)</dt><dd> 1&micro;L
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</dd><dt> KOD plus ver.2</dt><dd> 1&micro;L
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</dd><dt> MilliQ</dt><dd> 35&micro;L
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</dd><dt> Total</dt><dd> 50&micro;L
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</dd></dl>
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</li><li> Prepare control: instead of KOD plus ver.2, add 1&micro;L MilliQ.
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</li><li> Let stand for 2min at 94&#x2103;.
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</li><li> X cycles (1 cycle for 1kb) for 10s at 98&#x2103; and for Ymin (1min for 1kb) at 68&#x2103;.
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</li><li> Hold at 4&#x2103;.
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</li><li> Take 25&micro;L of the solutions into fresh tubes.
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</li><li> Add 1&micro;L <i>Dpn</i>I (10U/&micro;L).
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</li><li> Let stand for 1h at 37&#x2103;.
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</li><li> Agarose gel electrophoresis, using 5&micro;L of the solution for confirmation.
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</li><li> Mix the following.
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<dl><dt> Sample</dt><dd> 2&micro;L
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</dd><dt> Ligation high</dt><dd> 5&micro;L
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</dd><dt> T4 Polynucleotide Kinase (5U/&micro;L)</dt><dd> 1&micro;L
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</dd><dt> MilliQ</dt><dd> 7&micro;L
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</dd><dt> Total</dt><dd> 15&micro;L
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</dd></dl>
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</li><li> Let stand for 1h at 16&#x2103;.
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</li><li> Transformation, using 10&micro;L of the solution.
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</li></ol>
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<a name="PCR_Purification"></a><h5> <span class="mw-headline">PCR Purification</span></h5>
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<ol><li> Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
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</li><li> Add BufferPB about 5 times as much as the product of PCR.
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</li><li> Apply the solution to the column.
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</li><li> Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
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</li><li> Add 750&micro;L BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
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</li><li> Centrifuge for 1min and discard the through.
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</li><li> Centrifuge for additional 1min to remove residual buffer.
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</li><li> Place the column in a clean tube.
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</li><li> Add 10&micro;L BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
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</li><li> Centrifuge for 1min at 13000rpm.
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</li><li> Discard the column.
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</li></ol>
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</div>
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<!-- end of main -->
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Latest revision as of 02:26, 6 October 2011

Protocol

Listed below our protocol for this project.
We hope you find them useful!!

Cloning
Medium
Measurement

Retrieved from "http://2011.igem.org/Team:Kyoto/Protocol"