Edinburgh/Notebook/Gels

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Edinburgh iGem 2008

 

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
  • Gel 4:* minipreps M7 to M12, undigested
  • Gel 5:* minipreps M7 to M12, EcoRI/PstI
  • Gel 6:* minipreps M2, M3, M4, M6, HindIII; also P4 and P5 (fusion PCR)
  • Gel 7:* minipreps M10 and M11, EcoRI (two versions of this gel)
  • Gel 8:* PCR products P6 and P7 (mutagenic PCR on M11)
  • Gel 9:* maxipreps X1 and X2, etc.
  • Gel 10:* minipreps M13 to M18, E/P digests.
  • Gel 11:* minipreps M19 to M24, EcoRI digests, plus PCR P11.
  • Gel 12:* minipreps M25 to M30 of possible rbs+dxs clones: none look right.
  • Gel 13:* PCR P13 and P14 to mutated second EcoRI site on glgC clones M19 and M22.
  • Gel 14:* minipreps M31 to M42 (crtB and crtI clones) E/P, and P15 (rbs+appY)
  • Gel 15:* redigest of M32, M36 (crtB), M39, M40 (crtI) E/P; unsuccessful.
  • Gel 16:* purified PCR products for cenA, cenB, cenC, cex (P16, 17, 18, 19); unsuccessful.
  • Gel 17:* PCR products for cenA, cenB, cenC, cex (P20, 21, 22, 23) ~ second attempt; unsuccessful.
  • Gel 21:* P26 (rrnB from C fimi); unsuccessful.
  • Gel 22:* P27 (crtY ~ unsuccessful), P28, P29 (crtB, crtI ~ successful).
  • Gel 23:* P30 (repeat rrnB from C fimi) and PZNTA. Successful.
  • Gel 25:* P36 (glgC excision ~ successful), P37 (crtY ~ failed).
  • Gel 26:* P38~43 (cenA, cenB, cenC, cex from heat-killed cell suspension; cenA and cenB from 'impure' DNA solution.
  • Gel 27:* Repeat of Gel 26 but stained with SYBRsafe; putative cenA and cex fragments cut out.
  • Gel 28:* M49~54 (pSB1A2-rbs+crtI) digested with EcoRI.
  • Gel 29:* M55~60 (PZntA), M61~66 (CrtE+RBS) all digested with EcoRI and PstI.
  • Gel 30:* P46 (cenA), P47 (cex) from heat killed cell soln, annealing 65C. Failed. (30/07/08)
  • Gel 31*: M49 (X,SX,sac/spt), M50(X,SX,sac/spt), M63(E,EP), M67(E/P, sac1/spe1), P38(_cenA_). (30/07/08: OG)
  • Gel 32:* M55 to M60 double digestion with EcorI and PstI, and single digestion with EcoRI only. (30/07/08: Yan)
  • Gel 33:* M68~71 (pSB1A2-glgC m1+m2) digested with EcoRI+PstI, M72~75 (pSB1A2-rbs+dxs) digested with EcoRI, M76~79 (Babel2\+_crtE_) digested with EcoRI+PstI. See 01.08.2008 entry for results.
  • Gel 34:* M68~69 (pSB1A2-glgC m1+m2) digested with EcoRI alone and EcoRI+PstI, M72 (pSB1A2-rbs+dxs) digested with EcoRI alone and SacI+SpeI. See 01.08.2008 entry for results.
  • Gel 35:* P36 and L25. P36 successful, L25 yields clear glgC-length band but faint vector-length band.
  • Gel 36:* Double digestion of M80 to M96 with EcoRI and PstI, and PCR production of third mutagenesis of glgc mutant 1&2. Results were good for M80-82 (rbs\-_appY_\-pSB1A2), M84-85 (rbs\-_crtB_\-pSB1A2), M92 (_cex_\-pSB1A2) and P50 (_glgC_\-mut1,2+3). Other results were poor. (YAN&OG).
  • Gel 37:* Double digestion of M97 to M108 with EcoRI and PstI.
  • Gel 39:* Analytical digests using a) EcoRI and b) EcoRI/PstI of M70, M71 (both 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 to P58 (11.08.08: YAN)
  • Gel 42:* _C. fimi_ genomic DNA and PCR products P61~P63 from _C. fimi_ genomic DNA (11.08.08: CF)
  • Gel 43:* Double digests of BABEL2\-_crtE_ (M79) and Edinbrick1 with EcoRI and PstI, showing a single band at \~3kb for BABEL2\+_crtE_ and bands at \~2.5kb and 800bp for Edinbrick1. (11.08.08: AH)
  • Gel 44:* Double digests of BABEL1-glgc mut 1+2+3 (M117 to M120), pSB1A2-glgc mut 1+2 (M113-M116) and pSB1A2-CrtBI with EcoRI and PstI.(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)
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