Team:Osaka/week6
From 2010.igem.org
August 29 (Sun)
- Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
- Restriction digests
- for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
- for assembly: 2-4A, 3-11I with EcoRI, SpeI (upstream parts)
- Gel electrophoresis for confirmation
- Inserts seem to be present in all samples
- 3A assembly ligations:
- 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002
- 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
- Transformation of ligation products 002 and 003
August 30 (Mon)
- Restriction digests for 3A assembly
- 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
- 2-20J (CenA), 2-20H (Cex), F1 with XbaI, PstI
- Gel electrophoresis of the digests to confirm inserts
- all OK
- Transfer of 002 and 003 to solution culture (3 colonies each)
August 31 (Tue)
- Miniprep of 002, 003
- 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
- Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)
September 1 (Wed)
- Transformation (See Table 7)
- Miniprep of 5 separate cultures of 2-2O inoculated yesterday
- 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?
ID | Part Name | Resistance | Description |
---|---|---|---|
1-12D | <bbpart>BBa_E2030</bbpart> | K | yeast-optimized EYFP |
1-12B | <bbpart>BBa_E2020</bbpart> | K | yeast-optimized ECFP |
3-2K | <bbpart>BBa_K165001</bbpart> | A | yeast GAL1 promoter |
1-7D | <bbpart>BBa_J63006</bbpart> | A | yeast GAL1 promoter + Kozak sequence |
September 2 (Thu)
- Colony check
- 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
- 1-12B did not transform successfully
- 3A assembly ligations:
- 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
- 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
- Transformation of ligation products
September 3 (Fri)
- 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)
- Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
- all lengths ok
- Transfer of yesterday's transformations (colonies appeared later in the evening) to culture solution (2 colonies picked up from each plate)
September 4 (Sat)
- Miniprep of 004, 005
- Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
- 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 -> ??
- Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
- gel purification performed according to protocol in QIAquick Spin Handbook
- 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)