Purdue/8 July 2008

From 2008.igem.org

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Transformation, Take 3

Today, we attempted transformation of LacY (I763015) and LacZa (I732018) parts, along with a control pUC19 from Invitrogen. Yesterday, the two parts were prepped following the iGEM protocol (including the extra step). We used 2 spots of paper, and used 16uL of TE total per part (because the filter paper absorbs so much of it). We left them out overnight to let the DNA leak out of the filter paper for even longer. To start with, we used a NanoDrop UV-Vis spectrophotometer to see how much DNA was in each spot. Here are the results:

  • LacZa (I732018): 26.8 ng/uL
  • LacY (I763015): 43.1 ng/uL

For the actual transformation, we used 25uL of Invitrogen Max efficiency One Shot DH5a-T1 competent cells with 9uL of TE/DNA solution (all that we could get out the original 16uL). For the control, we added 5uL of pUC19 vector to 50uL of the same cells. For the rest of the transformation process, we followed the Invitrogen protocol that is supplied with the cells:

  • Mix DNA/Cells
  • Incubate on ice 30 min.
  • Heat shock 30s at 42C (no mixing or shaking)
  • Put on ice
  • Add 125uL SOC (for 25uL of cells) or 250uL SOC (for 50uL of cells)
  • Shake for 1 hr at 37C and 225 rpm
  • For Control:
    • Dilute 1:100
    • Plate 30uL on Amp plate
    • Incubate o/n at 37C
  • For Parts:
    • Plate 100uL on Amp plate
    • Incubate o/n at 37C

Edited by Janie Stine

In other news, the stabs of SOS promoter and LacZ genes came in today. We plated them on amp plates and let them grow o/n at 37C. The extra was placed at 4C.

Edited by Janie Stine