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

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

Sets of Primers that we used:  1.) Ligations A-I:
 * Distribute 9uL of "mastermix" into 0.2mL eppendorf tubes
 * Add 1uL DNA template (from cultured colonies)
 * 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