IGEM:IMPERIAL/2006/LabCalendar/2006-8-4

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Contents

Maxiprep

Maxiprep of the following parts:

  • 12D -> 24A (1)
  • 12D -> 24A (3)
  • 12D -> 24A (4)
  • 7A -> 3O (2)
  • 16P
  • 1I
  • 5M

Fusion PCR

No DNA was present on the gel.
Needs to be run again (after new oligos have arrived) to create new part using the freshly prepared maxi preps (5M). (New oligos have been ordered since previous ones contained errors in the sequence)

Testing of Maxipreps for DNA content

  • 5ul Maxi-prep DNA
  • 1ml water to blank

Stage 1 Ligation Assesment for 7A->30 (batch 2) and 12D->2H

  • 4 colonies of 7A->3O were isolated
  • 6 colonies of 12D->2H were isolated
  • Ran PCR on the colonies using the following primers:
    • For 7A->3O - C0062 2 and pBS1(A2/A3/AK3)
    • For 12D->2H - I13504 and pBS1(A2/A3/AK3)
  • Gel result to follow below
    • PCR product seen on colony 3 12D->2H
      • Some posibility of unspecific amplification due to prescence of two extra bands (see gel)
    • PCR product seen on colonies 2 and 3 7A->30 (batch 2)
      • Product from 2 is smaller than product from 3
  • As a result a conclusive answer as to whether the ligations were successful could not be reached
  • Colony 3 from 12D->2H and colonies 2 and 3 from 7A->3O (batch 2) were minprepped
    • Expect to see two bands if ligation successful
      • One band corresponding to plasmid
      • One band corresponding to insert (approx 800bp for both ligations)
    • Gels only showed one band corresponding to plasmid

Checking Maxipreps for DNA

  • Ran gels to check for DNA for (failed) maxipreps from 3/8/06 and maxipreps performed today.
  • DNA failed to stay in well
  • Used spectrophotometer instead


For spectrophotometer:
  • Use quartz cuvette
  • Take reading at 260nm
  • Use water as blank
  • 5ul Maxipreped DNA
  • Add DNA to 1ml water
  • Multiply the result by 10 to get []ug/ul
  • Required reading is 500ng
    • We doubled the amount of DNA to increase resolution.
    • Results: yesterday's maxipreps failed; today's worked:-)

Ligations

  • Digestions were successful this time - therefore we were able to ligate.
  • Parts ligated:
  • 12D->24A into 6B (twice - using different 12D->24A colonies)
  • 7A->30 into 9G
  • Riboswitch into 12D->24A

Electroporation

  • The part 4G from plate 2 was removed and electroporated and plated
  • Also all the ligated parts above were electroporated and plated
  • These plates should be collected on Saturday and placed in the cold room.
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