Team:Washington/Protocols/pGA

From 2011.igem.org

(Difference between revisions)
(→Gibson assembly efficiency assay)
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=Gibson assembly efficiency assay=
=Gibson assembly efficiency assay=
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'''Note: Prepare these mixtures on ice'''
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For each of the Gibson reactions, we added 20ng of gel-extracted PCR products for the inserts and backbones. For the pSB assay we used the primers for the inserts and () primers for the backbone.
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1. Obtain a 40 uL aliquot of BL21 cells.
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2. Add 120 uL of ice water to the aliquot.  
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3. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
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[[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]]
[[File:Washington_iGEM2011_pGAprotocol.png|thumb|right|175px| pGA vector Assembly ]]
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4. * INS + BCK tubes (x 2)
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'''Note: Prepare these mixtures on ice'''
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    * add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
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#. Obtain a 40 uL aliquot of BL21 cells.
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5. * INSctrl tube
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#. Add 120 uL of ice water to the aliquot.
 +
#. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
 +
#. * 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
     * add 100 pg of pLacGFP gel extract
     * add 100 pg of pLacGFP gel extract
6. * BCKctrl
6. * BCKctrl

Revision as of 20:40, 28 September 2011


Gibson assembly efficiency assay

For each of the Gibson reactions, we added 20ng of gel-extracted PCR products for the inserts and backbones. For the pSB assay we used the primers for the inserts and () primers for the backbone.

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.
  4. . * INS + BCK tubes (x 2)
      • add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
  5. . * INSctrl tube 
    * add 100 pg of pLacGFP gel extract

6. * BCKctrl 

    * add 100 pg of 1A3 gel extract


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)
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