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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=pGA Vector Assay=
 
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1. Prepare stocks for protease inhibitor, DNAse, and lysozyme.
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=pGA vector Assay=
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*a. Protease Inhibitor = 25x stock ; one tablet into 2ml of buffer
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*b. DNAses = 100x stock (10mg/ml initial → 0.1mg/mL final)
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*Prepare these mixtures on ice
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*c. Lysozyme = 10x stock (10mg/ml initial → 1mg/mL final)
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# Obtain a 40 uL aliquot of BL21 cells.
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2. Resuspend 50ml of each type of cell culture in 1ml of this mixture: 850ul sodium phosphate buffer, 40ul protease inhibitor, 10ul DNAse (0.1mg/ml final), and 100ul lysozyme (1mg/ml final).If testing multiple reactions, just multiply the volumes and divide into eppendorf tubes so that each reaction gets 1mL of cell suspension.
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# Add 120 uL of ice water to the aliquot.
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*a. Buffer: 1ml = 1000ul - (40+10+100) → 850ul 
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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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*b. Protease inhibitor: 25x (xml) = 1x (1ml) → 40ul
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#* INS + BCK tubes (x 2)
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*c. DNAses: (10mg/ml)Xul = (0.1mg/ml)1ml  → 10ul
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#** add 1 uL of 10X pGA Gibson product (1A3 BCK/INS pLacGFP)
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*d. Lysozyme: (1mg/ml)Xul = (1mg/ml) 1ml → 100ul
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#* INSctrl tube
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3. To lyse cells, add 111uL 10% triton (1% final) and mix the tube at room temperature for 30 min. Then, centrifuge the tubes at max. speed for 15 min (or until the liquid is very clear and not cloudy.
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#** add 100 pg of pLacGFP gel extract
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*x/(1000 +x) = 1/10  ….................. x = 111ul
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#* BCKctrl
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4. Transfer the liquid on top to new labeled eppendorf tube (leave the pellet of lysed stuff; our protein should be sufficiently soluble in aqueous solutions), tube perhaps containing master mix to initiate reaction.
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#** add 100 pg of 1A3 gel extract
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5. Place Target polyspring inserts into the Target DP glass vials.
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#Repeat steps 1-3 for the comparison vector pSB using the following instead:
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6. Cell Lysis
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#* 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
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*a. Sonicate --use micro tip, 60%, 30 on 30 off (POST-assay: cell lysate not so effective)
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#* 1 ng INS (pLacGFP- gel extract)
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*b. Bugbuster --Use 10x bugbuster, follow protocol (POST-assay: cell lysate not so effective)
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#* 1 ng BCK (T19-1A3- gel extract)
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*c. Triton X-100 --Use 1% triton (POST-assay: most effective for cell lysate)
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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 23:10, 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.
    • 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."