Team:Washington/Protocols/pGA

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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
    • 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
  1. 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)
  1. Once all the samples are ready, begin the transformation.
    • Rescue each sample in 500 mLs of LB
    • Incubate all samples @ 37oC for ~45 min.
  2. 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)