Team:Osaka/week6

From 2010.igem.org

August 29 (Sun)

1. Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
2. Restriction digests
   • for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
   • for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
3. Gel electrophoresis for confirmation
   • Inserts seem to be present in all samples
4. 3A assembly ligations:
   1. 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002
   2. 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as 003
   • 2-2O using XbaI, PstI digest from yesterday
   • 1-5A has Kan resistance
   • ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C
5. Transformation of ligation products 002 and 003

August 30 (Mon)

1. Restriction digests for 3A assembly
   • 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
   • 2-20J (CenA), 2-20H (Cex), F1 with XbaI, PstI
2. Gel electrophoresis of the digests to confirm inserts
   • all OK
3. Transfer of 002 and 003 to solution culture (3 colonies each)

August 31 (Tue)

1. Miniprep of 002, 003
2. Cut check of 002, 003 with EcoRI, SpeI
   • 003 was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in 003) also seemed to be longer than expected
3. Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)

September 1 (Wed)

1. Transformation (See Table 7)
2. Miniprep of 5 separate cultures of 2-2O inoculated yesterday
3. Cut check of 2-2O with XbaI, PstI
   • 0.7kbp bands in all 5 samples even though insert is supposed to be only 18bp - problem with the part (inconsistency confirmed from registry info page)
   • obtain Kozak sequence by PCR instead?

September 2 (Thu)

1. Colony check
   • 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
   • 1-12B did not transform successfully
2. 3A assembly ligations:
   1. 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
   2. 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
3. Transformation of ligation products

September 3 (Fri)

1. Colony check: yesterday's transformations seem to have failed; repeat of transformations of 004 and 005 with 50μl competent cells, 2μl ligation product (note: colonies appeared later; these repeats were then discarded)
2. Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
   • all lengths ok
3. Transfer of yesterday's transformations (colonies appeared later in the evening) to culture solution (2 colonies picked up from each plate)

September 4 (Sat)

1. Miniprep of 004, 005
2. Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
3. Gel electrophoresis
   • 1-7D -> OK
   • 004 -> insert length same as 001; since both upstream part 001 and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!
   • 005 -> ??
4. Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
   • gel purification performed according to protocol in QIAquick Spin Handbook
5. Ligation of gel-purified 001 to 1-2J or 2-22P, with vector 1-3A, to make 004 or 005 respectively (same 004 and 005 as designed before)

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