Team:UQ-Australia/Notebook/Experiment2

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==Miniprep Procedure==
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==Experiment 2==
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'''Production of Cleared Lysate'''
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'''Transformation of pSUTp. pG.pmt and pSUTp'''
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1. Pellet 1-10ml overnight cultures for 5 minutes
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The bacteria used for this transformation reaction was DH5α Max efficiency cells strain (chemical competent)
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pSUTp. pG. pmt  and pSUTp solution collected from 02/10/09 (click HERE fore further details)                            (Can you please put the link to 02/10.09 nanodrop result here?) were diluted to 10ng/uL solution to use for the transformation reaction.
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2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution
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'''Transformation control: pUC19'''
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*Click HERE for detailed protocol of transformation reaction.
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*While bacteria containing pSUTp.pG.pmt and pUC 19 were cultured on two separate Amp plates, bacteria with pSUTp was grown on Kan plate.
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*Incubate went overnight at 370C.
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*Results: Bacteria colonies were shown on plate in following morning
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*08/10/09: Pick isolate colonies from pSUTp and pSUTp.pG.pmt cultures.
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3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix
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Click HERE for detailed protocol of picking colonies.
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*4 colonies from pSUTp culture were picked and transfered into new tubes named pSUTp  Kan 1-4
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4. Add 10ul Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature
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*4 colonies from pSUTp.pG.pmt were picked and transferred into new tubes named  pSUTp.pG.pmt Amp 1-4
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NOTE: MerT and MerP primers arrived.
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5. Add 350 ul Neutralizer Solution; invert 4 times to mix
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6. Centrifuge at top speed for 10 minutes at room temperature.
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'''Binding of Plasmid DNA'''
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7. Insert Spin Column into Collection Tube
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8. Decant cleared lysate into Spin Column
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9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough and reinsert Column into Collection Tube.
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'''Washing'''
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10. Add 750ul Wash Solution (ethanol added). Centrifugeat top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube
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11. Repeat step 10 with 250ul Wash Solution
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12. Centrifuge at top speed for 2 minutes at room temperature
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'''Elution'''
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13. Transfer Spin Column to a sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column.
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14. Add 100ul of Nuclease-free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature.
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15. Discard column and store DNA at -20'C or below.
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Latest revision as of 12:27, 21 October 2009

Experiment 2

Transformation of pSUTp. pG.pmt and pSUTp

The bacteria used for this transformation reaction was DH5α Max efficiency cells strain (chemical competent) pSUTp. pG. pmt and pSUTp solution collected from 02/10/09 (click HERE fore further details) (Can you please put the link to 02/10.09 nanodrop result here?) were diluted to 10ng/uL solution to use for the transformation reaction.

Transformation control: pUC19

  • Click HERE for detailed protocol of transformation reaction.
  • While bacteria containing pSUTp.pG.pmt and pUC 19 were cultured on two separate Amp plates, bacteria with pSUTp was grown on Kan plate.
  • Incubate went overnight at 370C.
  • Results: Bacteria colonies were shown on plate in following morning
  • 08/10/09: Pick isolate colonies from pSUTp and pSUTp.pG.pmt cultures.

Click HERE for detailed protocol of picking colonies.

  • 4 colonies from pSUTp culture were picked and transfered into new tubes named pSUTp Kan 1-4
  • 4 colonies from pSUTp.pG.pmt were picked and transferred into new tubes named pSUTp.pG.pmt Amp 1-4

NOTE: MerT and MerP primers arrived.

Retrieved from "http://2009.igem.org/Team:UQ-Australia/Notebook/Experiment2"