Team:MIT gateway

From 2010.igem.org

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  <a href="#general">1 General Gateway Protocol</a><br>
  <a href="#general">1 General Gateway Protocol</a><br>
  <a href="#miniprep">2 Commented Protocol</a><br>
  <a href="#miniprep">2 Commented Protocol</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#virtual">2.1 Design PCR-Primers with attB1.1 and attB2.1 sites</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#virtual">2.1 Design PCR-Primers</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#digest">2.2 Gel Purify PCR Product</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#digest">2.2 Gel Purify PCR Product</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#antarctic">2.3 Confirm PCR Product with attB1/2.1 and DONR(TM)clone</a><br>
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  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#antarctic">2.3 Confirm PCR Product</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.4 Measure DNA</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#gel">2.4 Measure DNA</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#extract">2.5 Calculate PCR Product Needed</a><br>
  &nbsp;&nbsp;&nbsp;&nbsp;<a href="#extract">2.5 Calculate PCR Product Needed</a><br>
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</div></td><tr><td><br>
</div></td><tr><td><br>
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<div class="bodybaby" id="general">General Biobrick Construction Protocol</div><br>
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<div class="bodybaby" id="general">General Gateway Protocol</div><br>
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<b class="bolded" id="miniprep">Design PCR-Primers</b><br>
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<b class="bolded" id="miniprep">Miniprep.</b><br>
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<b class="bolded" id="miniprep">Gel Purify PCR Product</b><br>
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To extract DNA from cells, we used the QIAprep Spin Miniprep Kit according to protocol.<br><br>
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<b class="bolded" id="virtual">Virtual Restriction Mapping.</b><br>
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<b class="bolded" id="miniprep">Confirm PCR Product</b><br>
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The sequences of all our individual parts were entered into a shared Geneious library, and all of our Biobrick parts were created virtually parallel to actual construction. We used Geneious to find enzymes for restriction mapping and sequencing validation of parts.<br><br>
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<b class="bolded" id="digest">DNA Digestion.</b><br>
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<b class="bolded" id="miniprep">Measure DNA</b><br>
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For both restriction mapping and for digesting a plasmid to insert or extract a biobrick, the appropriate buffer was added 1:10 to a DNA sample and 10 units of the appropriate enzyme(s) were used per 1ug of DNA. The reactions were allowed to run at the temperature optimal for the enzyme for 1 hour, the enzymes were denatured at 65&deg;C for 20 minutes, and then the reactions were held at 16&deg;C.<br><br>
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<b class="bolded" id="antarctic">Antarctic Phosphatase.</b><br>
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<b class="bolded" id="miniprep">Calculate PCR Product Needed</b><br>
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To prevent recircularization of digested plasmids, we used NEB's Antarctic Phosphatase according to protocol.<br><br>
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<b class="bolded" id="gel">Gel Electrophoresis.</b><br>
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<b class="bolded" id="miniprep">Calculate volume of DONTR(TM) needed</b><br>
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To view lengths of DNA, Orange G 6x from NEB was added 1:3 to 300ng miniprepped (and sometimes digested) DNA and run against 5uL of Hyperladder I from NEB in the lanes of 1% UltraPure agarose (Invitrogen) gels in TAE buffer at 120V for 45 minutes.<br><br>
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<b class="bolded" id="extract">DNA Gel Extraction.</b><br>
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<b class="bolded" id="miniprep">Prepare Gateway Reaction</b><br>
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To extract the DNA from the agarose gel, the QIAquick Gel Extraction kit was used according to protocol.<br><br>
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<b class="bolded" id="miniprep">Retrieve 5x BP-Clonase II</b><br>
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<b class="bolded" id="miniprep">Add BP-Clonase to Gateway Reaction</b><br>
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<b class="bolded" id="miniprep">Incubate</b><br>
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 +
<b class="bolded" id="miniprep">Add ProteinaseK</b><br>
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 +
<b class="bolded" id="miniprep">Transform Bacteria</b><br>
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<b class="bolded" id="miniprep">Plate Bacteria</b><br>
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<div class="bodybaby" id="general">Commented Protocol</div><br>
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 +
<div class="bodybaby" id="general">Known Issues</div><br>
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<b class="bolded" id="transform">Transformation.</b><br>
 
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Aliquots of competent JM2.300 E.coli cells were thawed on ice for 8 minutes. Approximately 10ng of each plasmid were added to the competent cells and the tube was tapped to mix. The cells incubated on ice for 30 minutes after which they were heat shocked at 42&deg;C for exactly 30 seconds and then incubated on ice for 2 minutes. Each transformation received 0.9 mL of room temperature Super Optimal broth with Catabolite repression, and were shaken at 37&deg;C at 280 rpm for 60 minutes. On a LB plate with antibiotics that select for cells transformed with our DNA, 20 uL of the mixture was plated on one half, the rest of the cells were pelleted and plated on the other half. Plates incubated upright for 20 minutes then agar up for 12-16 hours at 37&deg;C.
 
<div class="bodybaby" id="cite">Citation</div><br>
<div class="bodybaby" id="cite">Citation</div><br>

Revision as of 21:33, 18 October 2010



bacterial biobrick construction

The Mammalian team used Gateway cloning to assemble its composite parts.

1 General Gateway Protocol
2 Commented Protocol
    2.1 Design PCR-Primers
    2.2 Gel Purify PCR Product
    2.3 Confirm PCR Product
    2.4 Measure DNA
    2.5 Calculate PCR Product Needed
    2.6 Calculate volume of DONRTM needed
    2.7 Prepare Gateway Reaction
    2.8 Retrieve 5x BP Clonase II
    2.9 Add BP-Clonase to Gateway Reaction
    2.10 Incubate
    2.11 Add ProteinaseK
    2.12 Transform Bacteria
    2.13 Plate Bacteria
3 Known Issues
4 Citation

General Gateway Protocol

Design PCR-Primers
Gel Purify PCR Product
Confirm PCR Product
Measure DNA
Calculate PCR Product Needed
Calculate volume of DONTR(TM) needed
Prepare Gateway Reaction
Retrieve 5x BP-Clonase II
Add BP-Clonase to Gateway Reaction
Incubate
Add ProteinaseK
Transform Bacteria
Plate Bacteria
Commented Protocol

Known Issues

Citation

1. Untergasser A. “Cloning – Gateway BP-Reaction II” Untergasser's Lab. Summer 2006. (include here the date when you accessed these page). .

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