Edinburgh/Notebook/Gels

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Gels

Gel 1: PCR reactions P1 (dxs), P2 (appY), P3 (glgC). (25.06.08)
Gel 2: EcoRI/PstI digests of M1 to M6 (pSB1A2+dxs). (30.06.08)
Gel 3: minipreps M2, M3, M4, M6, EcoRI, plus L1 and L2. (01.07.08)
Gel 4: minipreps M7 (BABEL1+glgC) and M8 to M12 (BABEL2+glgC), undigested. (01.07.08)
Gel 5: minipreps M7 to M12, EcoRI/PstI. (01.07.08)
Gel 6: minipreps M2, M3, M4, M6, HindIII; also P4 (BABEL1+glgC fusion PCR)and P5 (BABEL2+glgC fusion PCR). (01.07.08)
Gel 7: minipreps M10 and M11, EcoRI (two versions of this gel). (04.07.08)
Gel 8: PCR products P6 and P7 (mutagenic PCR on glgC [M11]). (04.07.08)
Gel 9: digests of maxipreps X1 (J33201) and X2 (pSB1A2+dxs). (08.07.08)
Gel 10: minipreps M13 to M18, EcoRI/PstI digests. (09.07.08)
Gel 11: M19 to M24 (BABEL2+glgC-mut2) EcoRI digests, plus PCR P11 (rbs+dxs). (10.07.08)
Gel 12: SacI/SpeI digests of M25 to M30 (possible rbs+dxs clones): none look right. Also shown is P12 (rbs+dxs fusion PCR product). (13.07.08)
Gel 13: PCR P13 and P14 to mutate out first EcoRI site from BABEL2+glgC-mut2 clones M19 and M22. (14.07.08)
Gel 14: minipreps M31 to M42 (pSB1A2+crtB and pSB1A2+crtI clones) EcoRI/PstI digests, and P15 (rbs+appY) (15.07.08)
Gel 15: redigest of M32, M36 (pSB1A2+crtB), M39, M40 (pSB1A2+crtI) with EcoRI/PstI; unsuccessful. (17.07.08)
Gel 16: purified PCR products for cenA, cenB, cenC, cex (P16 to P19); unsuccessful. (18.07.08)
Gel 17: PCR products for cenA, cenB, cenC, cex (P20 to P23); unsuccessful. (18.07.08)
Gel 18: EcoRI/PstI digests of M43 to M47 (BABEL2+glgC-mut1,2) and M48 (pSB1A2+rbs+dxs). M43 to M47 all look about right. M48 is ambiguous, could be incomplete deigestion, need to repeat. (19.07.08: CF)
Gel 19: P24 (cenA), P25 (cex) as well as SacI/SpeI digest of M48 (pSB1A2+rbs+dxs) and single (EcoRI) and double (EcoRI/PstI) digests of M36 (pSB1A2+crtB) and M42 (pSB1A2+crtI). (20.07.08: CF)
Gel 20: L15 (rbs+dxs with Edinbrick1) and L16 (rbs+appY with Edinbrick1) both failed. (22.07.08: OG, Yan)
Gel 21: P26 (rrnB from C. fimi); unsuccessful. (22.07.08: AM)
Gel 22: P27 (crtY, unsuccessful), P28 (pSB1A2+rbs+crtB, successful) and P29 (pSB1A2+rbs+crtI, successful). (23.07.08: AM, Yan, OG)
Gel 23: P30 (repeat rrnB from C. fimi) and pZntA, both successful. (23.07.08: AM)
Gel 24: Shows failure of P32-P35 (cenA, cenB, cenC and cex). (24.07.08: AM)
Gel 25: P36 (glgC-mut1,2 excision from BABEL2, successful), P37 (crtY, failed). (25.07.08: AH)
Gel 26: P38-41 (cenA, cenB, cenC, cex from heat-killed cell suspension) and P42-43 (cenA and cenB from 'impure' DNA solution). (28.07.08: AM)
Gel 27: Repeat of Gel 26 but stained with SYBRsafe. Putative cenA and cex fragments cut out. (29.07.08: AM)
Gel 28: M49-54 (pSB1A2+rbs+crtI) digested with EcoRI. (29.07.08: CF)
Gel 29: M55-60 (pSB1A2+pZntA), M61-66 (pSB1A2+rbs+CrtE) digested with EcoRI/PstI. Shows that ligations L23 and L24 (respectively) failed. (29.07.08: Yan, AM)
Gel 30: P46 (cenA), P47 (cex) from heat killed cell solution, annealing 65C. Gel shows failure of the PCR. (30/07/08)
Gel 31: P48 (crtY) and digests of M49 and M50 (pSB1A2-rbs+crtI) with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+crtE) with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+crtB) with a) EcoRI/PstI, b) sacI/speI. (30/07/08: OG)
Gel 32: M55-M60 (pSB1A2+pZntA) double digestion with EcoRI/PstI, and single digestion with EcoRI. (30.07.08: Yan)
Gel 33: M68-71 (pSB1A2+glgC-mut1,2) digested with EcoRI/PstI, M72-75 (pSB1A2+rbs+dxs) digested with EcoRI, M76-79 (BABEL2+crtE) digested with EcoRI/PstI. (01.08.2008: AM)
Gel 34: M68-69 (pSB1A2+glgC-mut1,2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2+rbs+dxs) digested with EcoRI alone and SacI/SpeI. (01.08.2008: AM)
Gel 35: P36 (glgC-mut1,2) and L25 (pSB1A2+glgC-mut1,2). P36 successful, L25 yields clear glgC-length band but faint vector-length band. (01.08.2008: AM)
Gel 36: P50 (glgC-mut1,2,3) and double digestion of M80-M96 with EcoRI/PstI. Results were good for M80-M82 (pSB1A2+rbs+appY), M84-M85 (pSB1A2+rbs+crtB), M92 (pSB1A2+cex) and P50. Other results were poor. (04.08.08: Yan, OG).
Gel 37: Double digestion of M97-M104 (pSB1A2+cenA) and M105-M108 (pSB1A2+cex) with EcoRI/PstI. Results were very poor apart from M97. (05.08.08: Yan)
Gel 38: Re-digestion of M97-M99 and M101 (all pSB1A2+cenA) and M105 and M108 (pSB1A2+cex) with a) EcoRI, b) EcoRI/PstI, and M92 (pSB1A2+cex) with a) PstI, b) EcoRI/PstI. (06.08.08: Yan) Gel 39: Analytical digests using a) EcoRI, b) EcoRI/PstI of M70-M71 (pSB1A2+glgC-mut1,2) and M92 (pSB1A2+cex). Results do not look good. Bands are wrong size to correspond to vector and no bands match an expected insert size. (06.08.08: CF)
Gel 40 PCR products of PcstA (P54) (and SOB). Results look promising (fairly prominent band <500bp). (07.08.08: CF)
Gel 41: PCR products of P55-P58 (cenA and cex): cex with KOD polymerase (P57) generated a single band of the desired size. However, cex with Velocity polymerase and cenA with both KOD or Velocity polymerase failed. (11.08.08: Yan)
Gel 42: PCR products P61-P63 (rrnB, cenA, cex): Very clear band for rrnB, no bands for either cenA or cex. (11.08.08: CF)
Gel 43: Double digests of BABEL2+crtE (M79) and Edinbrick1 with EcoRI/PstI, showing a single band at ~3kb for BABEL2+crtE digestion and bands at ~2.5kb and 800bp for Edinbrick1 digestion. (11.08.08: AH)
Gel 44: Double digests of M109-M112 (pSB1A2+crtB+crtI), M113-M116 (pSB1A2+glgC-mut1,2) and M117-M120 (pSB1A2+glgC-mut1,2,3). M110-M112 (pSB1A2+crtB+crtI) have a band at 2.5kb as expected. M109 (pSB1A2+crtB+crtI) gave a band at 3kb instead, so should be ignored from now on.(11.08.08: Yan)
Gel 45: P57 (cex from cells) EcoRI/PstI double digest, undigested P64 (cex from genomic DNA), P66 (zm1/ISA1) and P67 (zm2/ISA2). (12.08.08: CF/AM)
Gel 46: Double digests of M121 to M126: M121 to M123 (pSB1A2-CrtY-1 to pSB1A2-CrtY-3), M124 to M126 (pSB1A2-CrtY-RBS-1 to pSB1A2-CrtY-RBS-3) with EcoRI and PstI. (12.08.08: YAN)
Gel 47: P57 and P64 (cex) PstI digests and P65 (cenA) (12.08.08: AM)
Gel 48: M109 and M110 (_crtBI_) BamHI/EcoRV digest, M115 and M116 (pSB1A2\+_glgC_\-mut1,2) HindIII digest M115 and M116 (pSB1A2\+_glgC_\-mut1,2) EcoRI/HindIII digest. (13.08.08: AH)
Gel 49: P68 (cenB) and P69 (cenC) from _C. fimi_ genomic DNA. Failed. (13.08.08: AM)
Gel 50: M127~M138? Please confirm. (13.08.2008: OG, I believe, OG: i think this is the one when me andy and yan were loading it and taking pictures for the webpage.)
Gel 51: Undocumented gel stained with SYBR-safe with bands for purification?
Gel 52: P70 (glgC mut1+2), P71 (glgC mut 1+2+3). (14.08.2008: AM)
Gel 53: Minipreps of M139 to M143 (CrtE+PSB1A2) (15.08.08: YAN/OG)
Gel 54: P71A (glgC mut 1+2), P72 (glgC mut 1+2+3), P73~P77 (rrnB from _C. fimi_ genomic DNA) (15.08.08: AM)
Gel 55: M144-M147 (pSB1A2+cenA), M148-M151 (pSB1A2+cex) and M152-M155 (pSB1A2+dxs+crtE) digested with EcoRI/PstI. (18.08.08: AH)

Note: Remaining gels will be added when the relevant information is better organised.

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