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

• IGEM:IMPERIAL/2006/LabCalendar/2006-8-7 Events
• Day in the lab
• Project Progress

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