Team:Washington/Protocols/pGA

From 2011.igem.org

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{{Template:Team:Washington/Templates/Top}}
{{Template:Team:Washington/Templates/Top}}
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__NOTOC__
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=pGA vector Assay=
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=Gibson assembly efficiency assay=
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*Prepare this mixture on ice to prevent the reaction from beginning early
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=== PCR ===
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# Add 15uL of Gibson MasterMix to each tube.
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The PCR reactions were conducted at 20 μL volumes with 2x Phusion Flash polymerase master mix, 1 ng plasmid template, and 0.5 μM of each primer.  
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# Add ~20-50 ng of purified Backbone DNA to the reaction tube.  
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# Add ~20-50 ng of purified Insert DNA to the reaction tube.
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For the pGA assay, the insert came from a pGA1C3 vector and the insert came from a pGA1A3 vector expressing GFP. We used primers pGAprefix_fwd and pGAsuffix_rev to amplify the insert, and pGAsuffix_fwd and pGAprefix_rev to amplify the backbone.
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# Fill the remaining tubes with ice cold, distilled H20 to ensure a final volume of '''20 uL'''
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# After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.
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For the pSB assay, the insert came from a pSB3K3 vector and the insert came from a pSB1A3 vector expressing low GFP levels that are not strong enough to see visually. We used pre-existing Biobrick primers [http://partsregistry.org/wiki/index.php?title=Part:BBa_G1004 BioBrick_f] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_G1005 BioBrick_r] to amplify the insert and [http://partsregistry.org/wiki/index.php?title=Part:BBa_G1000 Suffix_f] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_G1001 Prefix_r] to amplify the backbone.
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<br/><br/><br/>
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=== Gibson reactions ===
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After performing PCR as outlined above, each fragment was run on a 1% agarose gel and gel-extracted using a Qiagen QIAquick gel extraction kit. 20 ng of gel-extracted insert and 20 ng of gel-extracted backbone were added to a 20 &mu;L Gibson reaction which was set up on ice and incubated at 50&deg;C for one hour.
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<br/><br/><br/>
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===pGA transformations===
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[[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]]
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'''Note: Prepare these mixtures on ice'''
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# Obtain a 40 uL aliquot of BL21 cells.
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# Add 120 uL of ice water to the aliquot.  
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# The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
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#* INS + BCK tubes (x 2)
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#** add 1 uL of 10x-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
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#* INSctrl tube
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#** add 100 pg of pLacGFP gel extract
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#* BCKctrl
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#** add 100 pg of 1A3 gel extract
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<br/><br/><br/><br/><br/><br/><br/>
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===pSB transformations===
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Repeat the process for the comparison pSB vector as follows:
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[[File:Washington_iGEM2011_pSBprotocol.png|thumb|right|175px| pSB vector Assembly ]]
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#* 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
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#* 1 ng INS (pLacGFP- gel extract)
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#* 1 ng BCK (T19-1A3- gel extract)
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#Once all the samples are ready, begin the [https://2011.igem.org/Team:Washington/Protocols/Elect. transformation].
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#* Rescue each sample in 500 mLs of LB
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#*Incubate all samples @ 37oC for ~45 min.
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#Plate 50 uL on each sample onto a LB + Chlor + Amp + IPTG plate.
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#*1 plate for each control in each vector set.  
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#*3 plates for ''each'' Gibson product tube in each set. ( 6 plates for each vector set + 2 control plates)

Latest revision as of 21:59, 28 September 2011


Gibson assembly efficiency assay

PCR

The PCR reactions were conducted at 20 μL volumes with 2x Phusion Flash polymerase master mix, 1 ng plasmid template, and 0.5 μM of each primer.

For the pGA assay, the insert came from a pGA1C3 vector and the insert came from a pGA1A3 vector expressing GFP. We used primers pGAprefix_fwd and pGAsuffix_rev to amplify the insert, and pGAsuffix_fwd and pGAprefix_rev to amplify the backbone.

For the pSB assay, the insert came from a pSB3K3 vector and the insert came from a pSB1A3 vector expressing low GFP levels that are not strong enough to see visually. We used pre-existing Biobrick primers BioBrick_f and BioBrick_r to amplify the insert and Suffix_f and Prefix_r to amplify the backbone.


Gibson reactions

After performing PCR as outlined above, each fragment was run on a 1% agarose gel and gel-extracted using a Qiagen QIAquick gel extraction kit. 20 ng of gel-extracted insert and 20 ng of gel-extracted backbone were added to a 20 μL Gibson reaction which was set up on ice and incubated at 50°C for one hour.


pGA transformations

pGA vector Assembly

Note: Prepare these mixtures on ice

  1. Obtain a 40 uL aliquot of BL21 cells.
  2. Add 120 uL of ice water to the aliquot.
  3. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
    • INS + BCK tubes (x 2)
      • add 1 uL of 10x-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
    • INSctrl tube
      • add 100 pg of pLacGFP gel extract
    • BCKctrl
      • add 100 pg of 1A3 gel extract








pSB transformations

Repeat the process for the comparison pSB vector as follows:

pSB vector Assembly
    • 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
    • 1 ng INS (pLacGFP- gel extract)
    • 1 ng BCK (T19-1A3- gel extract)
  2. Once all the samples are ready, begin the transformation.
    • Rescue each sample in 500 mLs of LB
    • Incubate all samples @ 37oC for ~45 min.
  3. Plate 50 uL on each sample onto a LB + Chlor + Amp + IPTG plate.
    • 1 plate for each control in each vector set.
    • 3 plates for each Gibson product tube in each set. ( 6 plates for each vector set + 2 control plates)
Retrieved from "http://2011.igem.org/Team:Washington/Protocols/pGA"