Edinburgh/Notebook/PCR products

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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)