Edinburgh/Notebook/PCR products

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Revision as of 13:30, 21 August 2008

Edinburgh iGem 2008

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 for rbs+dxs. Failed. (10.07.08)
P12: repeat fusion PCR for rbs+dxs, using L11 and primers rbs2fclon1+pSB1A2insr1. (13.07.08)
P13 and P14: MABEL mutation of site 1 on glgC mutant clones M19 and M22.
P15: fusion PCR for rbs+appY
P16, P17, P18 and P19: first attempts at cenA, cenB, cenC and cex.
P20, P21, P22 and P23: second attempts at cenA, cenB, cenC and cex.
P24, P25: repeat PCR for cenA and cex using Pfu. Failed.
P26: Positive control PCR with primers fd1 and rd1 to amplify 16S rRNA gene rrnB from Cellulomonas fimi.
P27: crtY from P. ananatis cells. - Failed
P28, P29: crtB and crtI from L18 and L19.
P30: Repeat positive control rrnB from C. fimi, annealing 1 minute.
P31: pZntA promoter
P32-P35: repeat PCR for cenA, cenB, cenC and cex, annealing 1 minute. 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.
P42~P45: cenA, cenB, cenC and cex from 'impure' DNA solution.
P46~P47: cenA and cex from 'impure' DNA solution, annealing 65C. Failed.
P48: crtY (new primer mixture) (previously P38a) (30.07.08: CF) - Failed
P49: Recreation of P12 (rbs+dxs) (previously P39a) (30.07.08: CF)
P50: third mutagenesis of glgc-mut1,2 (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 - Purified (07.08.08: CF)
P55: PCR products of CenA by KOD P56: PCR products of CenA by velocity P57: PCR products of CeX by KOD P58: PCR products of CeX by Velocity P59: pSB1A2-crtY (09.08.08: CF) P60: pSB1A2-rbs+crtY (09.08.08: CF) P61: 16s rRNA rrnB from C. fimi genomic DNA - successful(10.08.08: CF) P62: cenA from C. fimi genomic DNA - failed (10.08.08: CF) P63: cex from C. fimi genomic DNA - failed (10.08.08: CF) P64: cex from C. fimi genomic DNA, w/ glycerol, annealing 60°C. Success (11.08.08: AM) P65: cenA from C. fimi genomic DNA, w/ glycerol, annealing 56°C/20s. Success (12.08.08: AM) P66: zm1 (ISA1) (12.08.08: CF) P67: zm2 (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: zm1 (ISA1) from X11 (20.08.08: AM) P85: zm2 (ISA2) from X12 (20.08.08: AM)

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 '''P53:''' ''cex'' from heat killed ''C. fimi'' (07/08/08: YAN, AM) - Failed<br />
 '''P54:''' pCstA from ''E. coli'' cells - Purified (07.08.08: CF)<br />
+'''P55:''' PCR products of ''CenA'' by KOD
+'''P56:''' PCR products of ''CenA'' by velocity
+'''P57:''' PCR products of ''CeX'' by KOD
+'''P58:''' PCR products of ''CeX'' by Velocity
+'''P59:''' ''pSB1A2-crtY'' (09.08.08: CF)
+'''P60:''' ''pSB1A2-rbs+crtY'' (09.08.08: CF)
+'''P61:''' 16s rRNA rrnB from ''C. fimi'' genomic DNA - successful(10.08.08: CF)
+'''P62:''' ''cenA'' from ''C. fimi'' genomic DNA - failed (10.08.08: CF)
+'''P63:''' ''cex'' from ''C. fimi'' genomic DNA - failed (10.08.08: CF)
+'''P64:''' ''cex'' from ''C. fimi'' genomic DNA, w/ glycerol, annealing 60°C. Success (11.08.08: AM)
+'''P65:''' ''cenA'' from ''C. fimi'' genomic DNA, w/ glycerol, annealing 56°C/20s. Success (12.08.08: AM)
+'''P66:''' ''zm1'' (ISA1) (12.08.08: CF)
+'''P67:''' ''zm2'' (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:''' zm1 (ISA1) from X11 (20.08.08: AM)
+'''P85:''' zm2 (ISA2) from X12 (20.08.08: AM)