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Edinburgh/Notebook/PCR products
From 2008.igem.org
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<div id="header">{{Template:Team:Edinburgh/Templates/Header}}</div> | <div id="header">{{Template:Team:Edinburgh/Templates/Header}}</div> | ||
| + | '''[[Team:Edinburgh/Notebook|< Back to Notebook]]''' | ||
| + | |||
| + | == PCR Products == | ||
'''P1:''' ''dxs'' from ''Escherichia coli'' JM109. (25.06.08)<br /> | '''P1:''' ''dxs'' from ''Escherichia coli'' JM109. (25.06.08)<br /> | ||
'''P2:''' ''appY'' from ''E. coli'' JM109. (25.06.08)<br /> | '''P2:''' ''appY'' from ''E. coli'' JM109. (25.06.08)<br /> | ||
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'''P49:''' Recreation of P12 (rbs+''dxs'') (30.07.08: CF)<br /> | '''P49:''' Recreation of P12 (rbs+''dxs'') (30.07.08: CF)<br /> | ||
'''P50:''' Third mutagenesis of ''glgC''-mut1,2 by MABEL (04.08.08: Yan)<br /> | '''P50:''' Third mutagenesis of ''glgC''-mut1,2 by MABEL (04.08.08: Yan)<br /> | ||
| - | '''P51:''' | + | '''P51:''' P''cstA'' from ''E. coli'' cells. (06.08.08: CF) - Failed<br /> |
'''P52:''' ''cenA'' from heat killed ''C. fimi''. (07.08.08: Yan, AM) - Failed<br /> | '''P52:''' ''cenA'' from heat killed ''C. fimi''. (07.08.08: Yan, AM) - Failed<br /> | ||
'''P53:''' ''cex'' from heat killed ''C. fimi''. (07.08.08: Yan, AM) - Failed<br /> | '''P53:''' ''cex'' from heat killed ''C. fimi''. (07.08.08: Yan, AM) - Failed<br /> | ||
| - | '''P54:''' | + | '''P54:''' P''cstA'' from ''E. coli'' cells (07.08.08: CF)<br /> |
'''P55:''' ''cenA'' performed by KOD. (08.08.08: Yan)<br /> | '''P55:''' ''cenA'' performed by KOD. (08.08.08: Yan)<br /> | ||
'''P56:''' ''cenA'' performed by velocity. (08.08.08: Yan)<br /> | '''P56:''' ''cenA'' performed by velocity. (08.08.08: Yan)<br /> | ||
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'''P59:''' pSB1A2+''crtY'' (09.08.08: CF)<br /> | '''P59:''' pSB1A2+''crtY'' (09.08.08: CF)<br /> | ||
'''P60:''' pSB1A2+rbs+''crtY'' (09.08.08: CF)<br /> | '''P60:''' pSB1A2+rbs+''crtY'' (09.08.08: CF)<br /> | ||
| - | '''P61:''' 16s rRNA rrnB from ''C. fimi'' genomic DNA. (10.08.08: CF) - successful<br /> | + | '''P61:''' 16s rRNA ''rrnB'' from ''C. fimi'' genomic DNA. (10.08.08: CF) - successful<br /> |
'''P62:''' ''cenA'' from ''C. fimi'' genomic DNA. (10.08.08: CF) - Failed<br /> | '''P62:''' ''cenA'' from ''C. fimi'' genomic DNA. (10.08.08: CF) - Failed<br /> | ||
'''P63:''' ''cex'' from ''C. fimi'' genomic DNA. (10.08.08: CF) - Failed<br /> | '''P63:''' ''cex'' from ''C. fimi'' genomic DNA. (10.08.08: CF) - Failed<br /> | ||
'''P64:''' ''cex'' from ''C. fimi'' genomic DNA, with 10µl 50% glycerol, annealing 60°C. (11.08.08: AM) - Succesful<br /> | '''P64:''' ''cex'' from ''C. fimi'' genomic DNA, with 10µl 50% glycerol, annealing 60°C. (11.08.08: AM) - Succesful<br /> | ||
'''P65:''' ''cenA'' from ''C. fimi'' genomic DNA, w/ glycerol, annealing 56°C/20s. Success (12.08.08: AM)<br /> | '''P65:''' ''cenA'' from ''C. fimi'' genomic DNA, w/ glycerol, annealing 56°C/20s. Success (12.08.08: AM)<br /> | ||
| - | '''P66:''' '' | + | '''P66:''' ''SU1'' (12.08.08: CF)<br /> |
| - | '''P67:''' '' | + | '''P67:''' ''ISO2'' (ISA2) (12.08.08: CF)<br /> |
'''P68:''' ''cenB'' from ''C. fimi'' genomic DNA. Failed (13.08.08: AM)<br /> | '''P68:''' ''cenB'' from ''C. fimi'' genomic DNA. Failed (13.08.08: AM)<br /> | ||
'''P69:''' ''cenC'' from ''C. fimi'' genomic DNA. Failed (13.08.08: AM)<br /> | '''P69:''' ''cenC'' from ''C. fimi'' genomic DNA. Failed (13.08.08: AM)<br /> | ||
| - | '''P70:''' M43 (glgC mut 1+2). Failed (14.08.08: AM)<br /> | + | '''P70:''' M43 (''glgC'' mut 1+2). Failed (14.08.08: AM)<br /> |
| - | '''P71:''' M120 (glgC mut 1+2+3). Successful (14.08.08: AM)<br /> | + | '''P71:''' M120 (''glgC'' mut 1+2+3). Successful (14.08.08: AM)<br /> |
| - | '''P71A:''' Repeat M43 ( | + | '''P71A:''' Repeat M43 (''glgC'' mut 1+2). Succeessful (15.08.08: AM)<br /> |
| - | '''P72:''' Repeat M120 (glgC mut 1+2+3). Successful (15.08.08: AM)<br /> | + | '''P72:''' Repeat M120 (''glgC'' mut 1+2+3). Successful (15.08.08: AM)<br /> |
| - | '''P73~P77:''' rrnB from ''C. fimi'' genomic DNA purified on 14.08.08. P74 possibly successful (15.08.08: AM)<br /> | + | '''P73~P77:''' ''rrnB'' from ''C. fimi'' genomic DNA purified on 14.08.08. P74 possibly successful (15.08.08: AM)<br /> |
| - | '''P78A:''' rrnB from original C. fimi genomic DNA as positive control. Failed (15.08.08: AM)<br /> | + | '''P78A:''' ''rrnB'' from original ''C. fimi'' genomic DNA as positive control. Failed (15.08.08: AM)<br /> |
| - | '''P78:''' SOB2-glgC mut 1+2 from L45 (19.08.08: AM)<br /> | + | '''P78:''' SOB2-''glgC'' mut 1+2 from L45 (19.08.08: AM)<br /> |
| - | '''P79:''' SOB2-glgC mut 1+2+3 from L46 (19.08.08: AM)<br /> | + | '''P79:''' SOB2-''glgC'' mut 1+2+3 from L46 (19.08.08: AM)<br /> |
| - | '''P80:''' SOB2-glgC mut 1+2+3 from L47 (19.08.08: AM)<br /> | + | '''P80:''' SOB2-''glgC'' mut 1+2+3 from L47 (19.08.08: AM)<br /> |
| - | '''P81:''' SOB2-rbs- | + | '''P81:''' SOB2-rbs-''glgC'' mut 1+2 from L45 (19.08.08: AM)<br /> |
| - | '''P82:''' SOB2-rbs-glgC mut 1+2+3 from L46 (19.08.08: AM)<br /> | + | '''P82:''' SOB2-rbs-''glgC'' mut 1+2+3 from L46 (19.08.08: AM)<br /> |
| - | '''P83:''' SOB2-rbs-glgC mut 1+2+3 from L47 (19.08.08: AM)<br /> | + | '''P83:''' SOB2-rbs-''glgC'' mut 1+2+3 from L47 (19.08.08: AM)<br /> |
| - | '''P84:''' | + | '''P84:''' ''SU1'' (ISA1) from X11 (20.08.08: AM)<br /> |
| - | '''P85:''' | + | '''P85:''' ''ISO2'' (ISA2) from X12 (20.08.08: AM)<br /> |
| - | '''P86:''' | + | '''P86:''' ''SU1'' (ISA1) from X11, annealing 62°C (21.08.08: AM)<br/> |
| - | '''P87:''' | + | '''P87:''' ''ISO2'' (ISA2) from X12, annealing 62°C (21.08.08: AM)<br/> |
| - | '''P88:''' | + | '''P88:''' ''SU1'' (ISA1) from X11, annealing 60°C and glycerol (25.08.08: YAN)<br/> |
| - | '''P89:''' | + | '''P89:''' ''ISO2'' (ISA2) from X12, annealing 60°C and glycerol (25.08.08: YAN)<br/> |
| - | '''P90:''' CHU2268 - beta-glucosidase from cytophaga, annealing 55°C, extension 50s (26.08.08: YAN)<br/> | + | '''P90:''' CHU2268 ''bglX'' - beta-glucosidase from cytophaga, annealing 55°C, extension 50s (26.08.08: YAN)<br/> |
'''P91:''' Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob r1.(01.09.08:YAN)<br/> | '''P91:''' Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob r1.(01.09.08:YAN)<br/> | ||
'''P92:''' Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob.rbs.(01.09.08:YAN)<br/> | '''P92:''' Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob.rbs.(01.09.08:YAN)<br/> | ||
'''P93:''' Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob r1.(01.09.08:YAN)<br/> | '''P93:''' Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob r1.(01.09.08:YAN)<br/> | ||
'''P94:''' Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob.rbs.(01.09.08:YAN)<br/> | '''P94:''' Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob.rbs.(01.09.08:YAN)<br/> | ||
| - | '''P95:'' Site-directed mutagenesis of | + | '''P95:''' Site-directed mutagenesis of ''cex'', failed. (05.09.08: YAN)<br/> |
| - | '''P96:''' Repeat of Site-directed mutagenesis of | + | '''P96:''' Repeat of Site-directed mutagenesis of ''cex'' (11.09.08: YAN)<br/> |
| + | '''P99''' PCR for addition of rbs to ''glgC'' (CF, 24-9-8; OK)<br/> | ||
| + | '''P100''' PCR for affition of rbs to ''glgC16'' (CF, 24-9-8; OK)<br/> | ||
| + | '''P101''' PCR for addition of rbs to ''cex'' (CF, 4-10-8; looked OK but product later turned out to be wrong)<br/> | ||
| + | '''P102''' PCR for addition of rbs to ''cenA'' (CF, 4-10-8; looked OK but product later turned out to be wrong)<br/> | ||
| + | '''P105''' PCR of ''bglX'' with complete suffix (from ligation L74) to allow cloning EcoRI/PstI (CF, 9-10-8, OK)<br/> | ||
| + | '''P106''' mutagenic MABEL PCR to remove EcoRI site from ''SU1''. Failed. (CF)<br/> | ||
| + | '''P107''' repeat P106 with extended denaturation time and 10% v/v glucose: worked (CF, 10-10-8)<br/> | ||
| + | '''P108''' mutagenic MABEL PCR to remove EcoRI site from ''ISO2''. Extended denaaturation time but no glycerol. Failed. (CF, 11-10-8)<br/> | ||
| + | '''P109''' as P108 but with 10% v/v glycerol. Worked. (CF, 11-10-8)<br/> | ||
| + | '''P110''' repeat P101 (rbs+''cex''). Extended denaturation and 10% v/v glycerol. Worked. (CF, 22-10-8)<br/> | ||
| + | '''P111''' repeat P102 (rbs+''cenA''). Extended denaturation and 10% v/v glycerol. Worked. (CF, 22-10-8)<br/> | ||
| + | '''P112''' PCR for addition of rbs to ''bglX''. Worked. (CF, 23-10-8)<br/> | ||
| + | '''P113''' Fusion PCR on ligation L103 to make ''dxs-crtEBI''. Worked. (CF, 26-10-8)<br/> | ||
Latest revision as of 23:56, 29 October 2008
PCR Products
P1: dxs from Escherichia coli JM109. (25.06.08)
P2: appY from E. coli JM109. (25.06.08)
P3: glgC from E. coli JM109. (25.06.08)
P4: Fusion PCR for BABEL1+glgC. (01.07.08)
P5: Fusion PCR for BABEL2+glgC. (01.07.08)
P6: Mutagenic PCR to remove EcoRI site 1 from glgC (M11). (04.07.08)
P7: Mutagenic PCR to remove EcoRI site 2 from glgC (M11). (04.07.08)
P8: crtB (from Pantoea ananatis cells). (08.07.08)
P9: crtI (from Douglas's maxiprep of the mutated crtIB plasmid). (08.07.08)
P10: crtY (from P. ananatis cells). Failed. (08.07.08)
P11: Fusion PCR of rbs+dxs. Failed. (10.07.08)
P12: Repeat fusion PCR of rbs+dxs, using L11 and primers rbs2fclon1+pSB1A2insr1. (13.07.08)
P13-P14: MABEL mutation of site 1 on glgC-mut2 clones M19 and M22. (14.07.08)
P15: Rusion PCR for rbs+appY using L15 as a template. (15.07.08)
P16-P19: First attempts at cenA, cenB, cenC and cex. (17.07.08) - Failed
P20-P23: Second attempts at cenA, cenB, cenC and cex. (18.07.08) - Failed
P24-P25: Repeat PCR for cenA and cex using Pfu. (19.07.08) - Failed
P26: Positive control PCR with primers fd1 and rd1 to amplify 16S rRNA gene rrnB from C. fimi. (22.07.08: AM) - Failed
P27: crtY from P. ananatis cells. (23.07.08: AM, Yan, OG) - Failed
P28: Fusion PCR of rbs+crtB from L18. (23.07.08: AM, Yan, OG)
P29: Fusion PCR of rbs+crtI from L19. (23.07.08: AM, Yan, OG)
P30: Repeat positive control of rrnB from C. fimi, annealing 1 minute. (23.07.08: AM)
P31: pZntA promoter
P32-P35: Repeat PCR of cenA, cenB, cenC and cex, annealing 1 minute. (24.07.08: AM) - Failed
P36: M43 (glgC-mut1,2) with primers glgCf2/glgCr2. (25.07.08: AM)
P37: crtY from P. ananatis. (25.07.08: AM) - Failed
P38-P41: cenA, cenB, cenC and cex from heat-killed cell suspension. (28.07.08: AM)
P42-P45: cenA, cenB, cenC and cex from 'impure' DNA solution. (28.07.08: AM)
P46-P47: cenA and cex from 'impure' DNA solution, annealing 65C. (20.07.08: AM) - Failed
P48: crtY (new primer mixture) (30.07.08: CF) - Failed
P49: Recreation of P12 (rbs+dxs) (30.07.08: CF)
P50: Third mutagenesis of glgC-mut1,2 by MABEL (04.08.08: Yan)
P51: PcstA from E. coli cells. (06.08.08: CF) - Failed
P52: cenA from heat killed C. fimi. (07.08.08: Yan, AM) - Failed
P53: cex from heat killed C. fimi. (07.08.08: Yan, AM) - Failed
P54: PcstA from E. coli cells (07.08.08: CF)
P55: cenA performed by KOD. (08.08.08: Yan)
P56: cenA performed by velocity. (08.08.08: Yan)
P57: cex performed by KOD. (08.08.08: Yan)
P58: cex performed by Velocity. (08.08.08: Yan)
P59: pSB1A2+crtY (09.08.08: CF)
P60: pSB1A2+rbs+crtY (09.08.08: CF)
P61: 16s rRNA rrnB from C. fimi genomic DNA. (10.08.08: CF) - successful
P62: cenA from C. fimi genomic DNA. (10.08.08: CF) - Failed
P63: cex from C. fimi genomic DNA. (10.08.08: CF) - Failed
P64: cex from C. fimi genomic DNA, with 10µl 50% glycerol, annealing 60°C. (11.08.08: AM) - Succesful
P65: cenA from C. fimi genomic DNA, w/ glycerol, annealing 56°C/20s. Success (12.08.08: AM)
P66: SU1 (12.08.08: CF)
P67: ISO2 (ISA2) (12.08.08: CF)
P68: cenB from C. fimi genomic DNA. Failed (13.08.08: AM)
P69: cenC from C. fimi genomic DNA. Failed (13.08.08: AM)
P70: M43 (glgC mut 1+2). Failed (14.08.08: AM)
P71: M120 (glgC mut 1+2+3). Successful (14.08.08: AM)
P71A: Repeat M43 (glgC mut 1+2). Succeessful (15.08.08: AM)
P72: Repeat M120 (glgC mut 1+2+3). Successful (15.08.08: AM)
P73~P77: rrnB from C. fimi genomic DNA purified on 14.08.08. P74 possibly successful (15.08.08: AM)
P78A: rrnB from original C. fimi genomic DNA as positive control. Failed (15.08.08: AM)
P78: SOB2-glgC mut 1+2 from L45 (19.08.08: AM)
P79: SOB2-glgC mut 1+2+3 from L46 (19.08.08: AM)
P80: SOB2-glgC mut 1+2+3 from L47 (19.08.08: AM)
P81: SOB2-rbs-glgC mut 1+2 from L45 (19.08.08: AM)
P82: SOB2-rbs-glgC mut 1+2+3 from L46 (19.08.08: AM)
P83: SOB2-rbs-glgC mut 1+2+3 from L47 (19.08.08: AM)
P84: SU1 (ISA1) from X11 (20.08.08: AM)
P85: ISO2 (ISA2) from X12 (20.08.08: AM)
P86: SU1 (ISA1) from X11, annealing 62°C (21.08.08: AM)
P87: ISO2 (ISA2) from X12, annealing 62°C (21.08.08: AM)
P88: SU1 (ISA1) from X11, annealing 60°C and glycerol (25.08.08: YAN)
P89: ISO2 (ISA2) from X12, annealing 60°C and glycerol (25.08.08: YAN)
P90: CHU2268 bglX - beta-glucosidase from cytophaga, annealing 55°C, extension 50s (26.08.08: YAN)
P91: Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob r1.(01.09.08:YAN)
P92: Fusion PCR of P88 and SOB2 ligation products using Forward primer zm1 and Reverse primer sob.rbs.(01.09.08:YAN)
P93: Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob r1.(01.09.08:YAN)
P94: Fusion PCR of P89 and SOB2 ligation products using Forward primer zm2 and Reverse primer sob.rbs.(01.09.08:YAN)
P95: Site-directed mutagenesis of cex, failed. (05.09.08: YAN)
P96: Repeat of Site-directed mutagenesis of cex (11.09.08: YAN)
P99 PCR for addition of rbs to glgC (CF, 24-9-8; OK)
P100 PCR for affition of rbs to glgC16 (CF, 24-9-8; OK)
P101 PCR for addition of rbs to cex (CF, 4-10-8; looked OK but product later turned out to be wrong)
P102 PCR for addition of rbs to cenA (CF, 4-10-8; looked OK but product later turned out to be wrong)
P105 PCR of bglX with complete suffix (from ligation L74) to allow cloning EcoRI/PstI (CF, 9-10-8, OK)
P106 mutagenic MABEL PCR to remove EcoRI site from SU1. Failed. (CF)
P107 repeat P106 with extended denaturation time and 10% v/v glucose: worked (CF, 10-10-8)
P108 mutagenic MABEL PCR to remove EcoRI site from ISO2. Extended denaaturation time but no glycerol. Failed. (CF, 11-10-8)
P109 as P108 but with 10% v/v glycerol. Worked. (CF, 11-10-8)
P110 repeat P101 (rbs+cex). Extended denaturation and 10% v/v glycerol. Worked. (CF, 22-10-8)
P111 repeat P102 (rbs+cenA). Extended denaturation and 10% v/v glycerol. Worked. (CF, 22-10-8)
P112 PCR for addition of rbs to bglX. Worked. (CF, 23-10-8)
P113 Fusion PCR on ligation L103 to make dxs-crtEBI. Worked. (CF, 26-10-8)
