IGEM:IMPERIAL/2006/LabCalendar/2006-8-7

From OpenWetWare

Jump to: navigation, search


Contents

Minipreps

  • Miniprep to check ligations from Friday
  • A - 12D->24A->6B 2,1
  • B - "" 1,3
  • C - "" 2,2
  • D - "" 1,2
  • E - "" 1,4
  • F - "" 1,6
  • G - "" 1,5
  • H - "" 2,3
  • I - "" 1,1
  • J - Riboswitch ->12D->24A 8 (via PCR)
  • K - "" 1(via PCR)
  • L - "" 2(via PCR)
  • M - "" 3(via PCR)
  • N - "" 4(via PCR)
  • O - "" 5(via PCR)
  • P - "" 6(via PCR)
  • Q - "" 7(via PCR)
  • R - 7A->3O->9G 1
  • S - "" 2
  • T - "" 3
  • U - "" 4
  • V - "" 5
  • W - "" 6
  • X - "" 7
  • Y - "" 9
  • Z - "" 8
  • AA - "" 10
  • Some confirmed successful
  • Recultured A, R, T, and W (?) for maxiprep tomorrow

Ligation

  • Redo ligation 12D->2H
  • In digestion, saw two bands, cut them both out and have tried both of them
  • We'll see which one gives the better result (hopefully the correct result)
  • Ligation was left overnight
  • In glass milk purification, did not do the second 65C step, hopefully will not affect results
  • Electroporation tomorrow morning

Screening stage 2 ligations by PCR

  • The 27 colonies above were additionally screened by PCR
  • For PCR:
    • "Mastermix" of 100ul for each of the three ligations:
      • 47uL MiliQ
      • 20uL MgCl2 (25mM)
      • 10uL Taq buffer (NH4)2SO4-MgCl2
      • 5uL dNTP mix (10mM)
      • 2uL Primer I (for primers used see below)
      • 2uL Primer II
      • 2uL Taq Polymerase
  • Distribute 9uL of "mastermix" into 0.2mL eppendorf tubes
  • Add 1uL DNA template (from cultured colonies)


Sets of Primers that we used:
1.) Ligations A-I:

  • pSB1(A2/A3/AK3) Tm = 56.8 C
  • I13504 Tm = 51.9 C

2.) Ligations J-Q:

  • Riboswitch Primer Tm = 44.2 C
  • ...2Reverse Primer (Riboswitch) Tm= 61.0 C

3.) Ligations R-AA:

  • pSB1(A2/A3/AK3) Tm = 56.8 C
  • C00622 Tm = 43.6 C


PCR Program:
1.) Ligations A-I: J37015 No2

  • 30 cycles
    • 94 C for 1min.
    • 46.9 C for 1min. (Annealing Temperature)
    • 72 C for 40sec
    • Final Ext.: 72 C for 10min.
    • Final Hold: 4 C

2.) Ligations J-Q: Riboswitch

  • same as above but Annealing Temp.: 44.2 C

3.) Ligations R-AA: J37019 No2

  • same as above but Annealing Temp.: 38.6 C


After PCR: Run gels to check whether PCR & ligations were successful

  • Add 2uL loading buffer to each PCR eppendorf tube
    • Note: Some eppendorfs strangely had only little liquid inside after PCR: C,E,H,I,K,Q
  • Gel: 90V for 30min.


Results:

  • PCR gel does not give the same results as miniprep, there seems to be no correlation
  • Photos of the gel to be uploaded
  • Gel for Ligations 3.) has not been run yet - needs to be run tomorrow.

Fusion PCR

ran one of the 2 reactions, ran out of nucleotieds, couldn't do second one. Tubes frozen Testing tomorrow

Personal tools