Team:Washington/Protocols/pGA

From 2011.igem.org

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=pGA vector Assay=
=pGA vector Assay=
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*Prepare this mixture on ice to prevent the reaction from beginning early
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*Prepare these mixtures on ice
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# Obtain a 40 uL aliquot of BL21 cells.
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# Add 15uL of Gibson MasterMix to each tube.
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# Add 120 uL of ice water to the aliquot.  
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# Add ~20-50 ng of purified Backbone DNA to the reaction tube.  
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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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# Add ~20-50 ng of purified Insert DNA to the reaction tube.
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#* INS + BCK tubes (x 2)
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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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#** add 1 uL of 10X pGA Gibson product (1A3 BCK/INS pLacGFP)
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# After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.
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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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#Repeat steps 1-3 for the comparison vector pSB using the following instead:
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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)

Revision as of 21:58, 27 September 2011


pGA vector Assay

  • 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 pGA Gibson product (1A3 BCK/INS pLacGFP)
    • INSctrl tube
      • add 100 pg of pLacGFP gel extract
    • BCKctrl
      • add 100 pg of 1A3 gel extract
  4. Repeat steps 1-3 for the comparison vector pSB using the following instead:
    • 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
    • 1 ng INS (pLacGFP- gel extract)
    • 1 ng BCK (T19-1A3- gel extract)
  5. Once all the samples are ready, begin the transformation.
    • Rescue each sample in 500 mLs of LB
    • Incubate all samples @ 37oC for ~45 min.
  6. 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"