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Edinburgh/Notebook/Minipreps
From 2008.igem.org
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'''M217 to M221:''' dxs+crtEBI. (15.09.08: AM)<br/> | '''M217 to M221:''' dxs+crtEBI. (15.09.08: AM)<br/> | ||
'''M222 to M225:''' Plac+dxs+LIMS1+appY patches 1, 2, 6 and 7 (CF, 23-9-8). All look OK on gel. M222 confirmed by sequencing.<br/> | '''M222 to M225:''' Plac+dxs+LIMS1+appY patches 1, 2, 6 and 7 (CF, 23-9-8). All look OK on gel. M222 confirmed by sequencing.<br/> | ||
| - | '''M226 to M229:''' SOB-ZmISA1. (CF, 27-9-8) Only M229 looks plausible on gel. <br/> | + | '''M226 to M229:''' SOB-ZmISA1. (CF, 27-9-8) Only M229 looks plausible on gel, but sequencing shows problem at end. <br/> |
'''M230 to M232:''' SOB-ZmISA2. (CF, 27-9-8) All looked OK; sequencing showed M230 and 232 OK, 231 problem with suffix.<br/> | '''M230 to M232:''' SOB-ZmISA2. (CF, 27-9-8) All looked OK; sequencing showed M230 and 232 OK, 231 problem with suffix.<br/> | ||
'''M233 to M235:''' pSB1A2-cex mutant (CF) Sequence showed M233 OK.<br/> | '''M233 to M235:''' pSB1A2-cex mutant (CF) Sequence showed M233 OK.<br/> | ||
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'''M244 to M245:''' pSB1A2-rbs-glgC16 (CF, 28-9-8)<br/> | '''M244 to M245:''' pSB1A2-rbs-glgC16 (CF, 28-9-8)<br/> | ||
'''M246 to M247:''' pSB1A2-bglX (CF, 28-9-8). M247 looks plausible but sequencing shows wrong. <br/> | '''M246 to M247:''' pSB1A2-bglX (CF, 28-9-8). M247 looks plausible but sequencing shows wrong. <br/> | ||
| + | '''M248 to M253:''' pSB1A2-crtBI-dxs-crtE (CF, 30-9-8). All look wrong.<br/> | ||
| + | '''M254 to M255:''' pSB1A2-Plac-glgC (CF, 2-10-8). Look OK. M254 confirmed by sequencing.<br/> | ||
| + | '''M256 to M257:''' pSB1A2-Plac-glgC16 (CF, 2-10-8). Look OK. M256 confirmed by sequencing.<br/> | ||
| + | '''M258:''' pSB1A2-PcstA-lacZ' (CF, 2-10-8). Looks OK, confirmed by sequencing.<br/> | ||
| + | '''M259:''' pSb1A2-PcstA-xylE (CF, 2-10-8). Looks OK, confirmed by sequencing.<br/> | ||
| + | '''M260 to M265:''' SOB-ZmISA1 (CF, 3-10-8). M265 looks OK on gel, confirmed by sequencing. | ||
| + | '''M266 to M269:''' SOB-ZmISA2 (CF, 4-10-8). M269 looks plausible on gel. | ||
| + | '''M270 to M272:''' pSB1A2+rbs+cex (CF, 8-10-8). All look wrong.<br/> | ||
| + | '''M273 to M275:''' pSB1A2+rbs+cenA (CF, 8-10-8). M273, 274 looked OK but turned out to be wrong gene.<br/> | ||
| + | '''M276 to M278:''' pSB1A2+rbs+cex (CF, 11-10-8). M276 looked OK but turned out to be wrong gene.<br/> | ||
| + | '''M279 to M282:''' pSB1A2+bglX (CF, 14-10-8). All looked Ok on gel, but sequencing showed that all but M281 had frameshift mutations in a run of A near the start resulting in premature termination. M281 seems OK though.<br/> | ||
| + | '''M283 to M286:''' from mutagenesis of ZmISA1 (CF, 14-10-8 and 15-10-8). M286 looks wrong, others still seem to have the EcoRI site.<br/> | ||
| + | '''M287 to M290:''' from mutagenesis of ZmISA2 (CF, 15-10-8). All seem to still have EcoRI site.<br/> | ||
Revision as of 17:17, 29 October 2008
Minipreps of Confirmed Clones
- M2: pSB1A2+dxs
- M15: pSB1A2+appY
- (?) M36: pSB1A2+crtB (sequencing results not recorded, but follow-up did occur)
- (?) M42: pSB1A2+crtI (sequencing results not recorded, but follow-up did occur)
- M43: BABEL2(?)+glgC double mutant.
- M50: pSB1A2+rbs+crtI
- M63: BABEL2+rbs+crtE
- M67: pSB1A2+rbs+crtB
- M72: pSB1A2+rbs+dxs
- M79: BABEL2+crtE
- M82: pSB1A2+rbs+appY
- M109: pSB1A2+crtBI
- (?) M121: pSB1A2+CrtY (sequencing results not posted; no follow-up recorded)
- (?) M124 pSB1A2+rbs+CrtY (sequencing results not posted; no follow-up recorded)
- M116: pSB1A2+glgC double mutant
- M130: pSB1A2+PcstA
- M137: pSB1A2+dxs+lims
- M150: pSB1A2+cex
- M152: pSB1A2+dxs+crtE
- M154: not sequenced, but similar to M152.
- M155: not sequenced, but also similar to M152; ligated to M110 (CrtBI) in L62.
- M162: pSB1A2+dxs+lims+appY
- M212: pSB1A2+glgC triple mutant (ie, glgC16)
- M222: pSB1A2+Plac+dxs+LIMS1+appY
- M233: pSB1A2+cex mutant (PstI site removed)
- M234, 235: not sequenced, but similar to M233.
- M237, 238: pSB1A2+cenA
- M243: pSB1A2+rbs+glgC
- M245: pSB1A2+rbs+glgC16
- M254: pSB1A2+Plac+rbs+glgC
- M256: pSB1A2+Plac+rbs+glgC16
- M258: pSB1A2+PcstA+lacZ' (BBa_J15202)
- M259: pSB1A2+PcstA+xylE (BBa_J33204)
- M281: pSB1A2+bglX coding sequence
- M303: pSB1A2+rbs+cex
- M305: pSB1A2+rbs+cenA
- M307: pSB1A2+rbs+bglX
Complete List of Minipreps
M1 to M6: pSB1A2+dxs transformants (plate 11). M2, M3, M4 and M6 all looked correct on gel 2. (30.06.08) - M2 was sequenced to confirm identity and was maxiprepped as X2.
M7: BABEL1+glgC transformant (plate 12). Did not look right on gel. (30.06.08)
M8 to M12: BABEL2+glgC transformants (plate 13). M10 and M11 both looked good on a gel, but sequencing showed that both had the insert in the reverse orientation. (30.06.08)
M13 to M18: pSB1A2+appY transformants. Gel 10 showed that M15 to M18, and possibly M14, all seemed to have inserts the right size. (09.07.08)
M19 to M24: BABEL2+glgC clones after mutation of EcoRI site 2. EcoRI digests on *Gel 11* suggest that *M19, M21 and M22* may have lost the EcoRI site. (10.07.08)
M25 to M30: possible rbs+dxs transformants from plate 34. (13.07.08)
M31 to M36: pSB1A2+crtB clones from plate 35. (14.07.08)
M37 to M42: pSB1A2+crtI clones from plate 36. (14.07.08)
M43 to M47: possible BABEL2+glgC double mutants (both EcoRI sites gone). (19.07.08)
M48: a possible pSB1A2+rbs+dxs clone. (19.07.08)
M49 to M54: pSB1A2-rbs+crtI. (29.07.08: CF)
M55 to M60: Putative pSB1A2+pZntA (L23). (29.07.08: Yan, Nimisha) - M50 sequenced (31.07.08: CF)
M61 to M66: Putative pSB1A2+rbs+crtE (L22). (29.07.08: Yan, Nimisha) - M63 sequenced (31.07.08: CF)
M67: rbs+crtB (L21) (29.07.08: Yan, Nimisha) - Sequenced (31.07.08: CF)
M68 to M71: pSB1A2+glgC-mut1,2 from plate 73 (31.07.08: AM, Yan)
M72 to M75: pSB1A2+rbs+dxs from plate 74 (31.07.08: AM, Yan)
M76 to M79: BABEL2+crtE from plate 74 (31.07.08: AM, Yan)
M80 to M83: pSB1A2+rbs+appY from plate 83. (04.08.08: Yan, OG)
M84 to M85: pSB1A2+rbs+crtB from plate 84. (04.08.08: Yan, OG)
M86 to M90: pSB1A2+cenA from plate 85. (04.08.08: Yan, OG)
M91 to M96: pSB1A2+cex from plate 86. (04.08.08: Yan, OG)
M97 to M104: pSB1A2+cenA from plate 85. (05.08.08: Yan)
M105 to M108: pSB1A2+cex from plate 85. (05.08.08: Yan)
M109 to M112: pSB1A2+crtB+crtI and plate 102 (11.08.08: OG)
M113 to M116: pSB1A2+glgC-mut1,2 and plate 103 (11.08.08: OG)
M117 to M120: pSB1A2+glgC-mut1,2,3 and plate 94 (11.08.08: OG)
M121 to M123: pSB1A2-crtY (12.08.08: OG)
M124 to M126: pSBA12-rbs+crtY (12.08.08: OG)
M127 to M130: pSB1A2-PcstA from plate 115 (13.08.08: OG)
M131 to M134: Minipreps of dxs+crtE (1,2,3,4) Note: M132 might be M136 (13.08.08: OG)
M135 to M138: Minipreps of dxs+lims (1,2,3,4) Note: M136 might be M132 (13.08.08: OG)
M139 to M143: Minipreps of pSB1A2 +CrtE (1 to 4) Note: M142 and M143 both made from culture 4.(15.08.08: YAN/OG)
M144 to M147: pSB1A2+cenA from plate 129 - Methanol used in preparation rather than ethanol! (18.08.08: OG)
M148 to M151: pSB1A2+cex from plate 129. (18.08.08: OG)
M152 to M155: pSB1A2+dxs+crtE from plate 130 (18.08.08: OG)
M156 to M159: pSB1A2+cenA (20.08.08: OG)
M160 to M163: Plate 155 streaks 1-4 (L48, pSB1A2+dxs+lims+appY) (25.08.08: AH)
M164: Plate 155 streak 5 (L49, pSB1A2+crtB+crtI+appY) (25.08.08: AH)
M165 to M168: Plate 158 streaks 1-4 (L50, SOB+glgC-mut1,2) (25.08.08: AH)
M169 to M172: Plate 159 streaks 1-4 (L51, SOB+glgC-mut1,2,3) (25.08.08: AH, Yan)
M173: Plate 160 streak 1 (L55 (SOB+rbs+glgC-mut1,2,3) (25.08.08: Yan)
M174 to M175: Plate 160 streaks 2 and 3 (L52, SOB+glgC-mut1,2,3) (25.08.08: Yan)
M176 to M178: Plate 161 streaks 1-3 (L53, SOB+rbs+glgC-mut1,2) (25.08.08: Yan)
M179 to M181: Plate 163 streaks 1-3 (L56, pSB1A2+pCstA+lacZ') (25.08.08: Yan)
M182 to M187: pSB1A2+CenA (01.09.08: YAN)
M188 to M193: CrtBI+AppY (01.09.08: YAN)
M194 to M197: AppY+dxs+Lims (02.09.08:YAN)
M198 to M200: L58 = self-ligation of P91 (SOB2-zm1). M199 and M200 were missed out by accident. (09.09.08: Yan)
M201 to M203: L59 = self-ligation of P92 (SOB2-rbs-zm1). (09.09.08: Yan)
M204 to M206: L60 = self-ligation of P93 (SOB2-zm2). (09.09.08: Yan)
M207 to M209: L61 = self-ligation of P94 (SOB2-rbs-zm2). (09.09.08: Yan)
M210 to M212: pSB1A2-glgc triple mutants. (15.09.08: AM)
M213: dxs+CrtEBI. (15.09.08: AM)
M214: pSB1A2-CenA. (15.09.08: AM)
M215 to M216: pSB1A2-bglX. (15.09.08: AM)
M217 to M221: dxs+crtEBI. (15.09.08: AM)
M222 to M225: Plac+dxs+LIMS1+appY patches 1, 2, 6 and 7 (CF, 23-9-8). All look OK on gel. M222 confirmed by sequencing.
M226 to M229: SOB-ZmISA1. (CF, 27-9-8) Only M229 looks plausible on gel, but sequencing shows problem at end.
M230 to M232: SOB-ZmISA2. (CF, 27-9-8) All looked OK; sequencing showed M230 and 232 OK, 231 problem with suffix.
M233 to M235: pSB1A2-cex mutant (CF) Sequence showed M233 OK.
M236 to M238: pSB1A2-cenA (CF, 27-9-8) . M236 clearly wrong on gel, others plausible. Sequence showed M237 OK.
M239 to M241: pSB1A2-bglX (CF, 27-9-8) Only 240 looked plausible on gel, sequence showed wrong.
M242 to M243: pSB1A2-rbs-glgC (CF, 28-9-8)
M244 to M245: pSB1A2-rbs-glgC16 (CF, 28-9-8)
M246 to M247: pSB1A2-bglX (CF, 28-9-8). M247 looks plausible but sequencing shows wrong.
M248 to M253: pSB1A2-crtBI-dxs-crtE (CF, 30-9-8). All look wrong.
M254 to M255: pSB1A2-Plac-glgC (CF, 2-10-8). Look OK. M254 confirmed by sequencing.
M256 to M257: pSB1A2-Plac-glgC16 (CF, 2-10-8). Look OK. M256 confirmed by sequencing.
M258: pSB1A2-PcstA-lacZ' (CF, 2-10-8). Looks OK, confirmed by sequencing.
M259: pSb1A2-PcstA-xylE (CF, 2-10-8). Looks OK, confirmed by sequencing.
M260 to M265: SOB-ZmISA1 (CF, 3-10-8). M265 looks OK on gel, confirmed by sequencing.
M266 to M269: SOB-ZmISA2 (CF, 4-10-8). M269 looks plausible on gel.
M270 to M272: pSB1A2+rbs+cex (CF, 8-10-8). All look wrong.
M273 to M275: pSB1A2+rbs+cenA (CF, 8-10-8). M273, 274 looked OK but turned out to be wrong gene.
M276 to M278: pSB1A2+rbs+cex (CF, 11-10-8). M276 looked OK but turned out to be wrong gene.
M279 to M282: pSB1A2+bglX (CF, 14-10-8). All looked Ok on gel, but sequencing showed that all but M281 had frameshift mutations in a run of A near the start resulting in premature termination. M281 seems OK though.
M283 to M286: from mutagenesis of ZmISA1 (CF, 14-10-8 and 15-10-8). M286 looks wrong, others still seem to have the EcoRI site.
M287 to M290: from mutagenesis of ZmISA2 (CF, 15-10-8). All seem to still have EcoRI site.
