Team:Washington/Protocols/pGA

From 2011.igem.org

(Difference between revisions)
(→pGA vector Assay)
(→pGA vector Assay)
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*Prepare these mixtures on ice
*Prepare these mixtures on ice
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# Obtain a 40 uL aliquot of BL21 cells.
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1. 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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2. 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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3. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.  
[[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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#* INS + BCK tubes (x 2)
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4. * 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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** add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
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#* INSctrl tube
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5. * INSctrl tube
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#** add 100 pg of pLacGFP gel extract
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** add 100 pg of pLacGFP gel extract
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#* BCKctrl
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6. * BCKctrl
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#** add 100 pg of 1A3 gel extract
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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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7. Repeat steps 1-3 for the comparison vector pSB using the following instead:
[[File:Washington_iGEM2011_pSBprotocol.png|thumb|right|175px| pSB vector Assembly ]]
[[File:Washington_iGEM2011_pSBprotocol.png|thumb|right|175px| pSB vector Assembly ]]
#* 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
#* 1 uL of pSB Gibson product (T19-1A3/pLacGFP)

Revision as of 07:18, 28 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.

pGA vector Assembly

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

7. Repeat steps 1-3 for the comparison vector pSB using the following instead:

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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