IGEM:Peking/2007/Switch-Notebook/2007-7-9

From OpenWetWare

Jump to: navigation, search

Experimenter: NM & LCB

Contents

Contents

Precipitate with ethanol: pCC002^^ (C/N), SDD’-EGFP (B/C)

  • Time: 8:30am

NotI不能乙醇失活!! 必须用苯酚抽提失活, 呜呜… 做错了

重切pCC002^^ (C/N)!

Image:PKU Switch 07-7-9 pCC002^^(NotI ClaI) 7.8 7.9 SDD SDD'.gif

l to r: pCC002 (wrong), pCC002 (right), none, SDD', SDD, Marker

KOD PCR of SDD-EGFP with temperature gradient

  • Primers: SDD-EGFP-BamHI-F, GFP-ClaI-R
  • Template: SDD-EGFP-pMD18T

Image:PKU Switch 07-7-9 SDD EGFP temperature gradient.gif

l to r: SDD-EGFP(1-4), Marker

Send for sequencing: SD1(1-3), SD2(1-3)

Transformation of ECFP-pCC002

Digestion tests

  • Single digestion test of pLX007(1,2) with SalI/XhoI
  • Double digestion test of lacZa-pMD18-T with SalI/XhoI

Image:PKU Swtich 07-7-9 LacZa(1-3) pLX007 salI(1-2) xhoI(1-2).gif

l to r: lacZa(1-3), pLX007^ SalI1-2, pLX007^ XhoI1-2, Marker


Double digestion of pLX007(1) with SalI/XhoI

Double digestion of lacZa-pMD18-T(2) with SalI/XhoI

Double digestion of SDD-EGFP with BamHI & ClaI

PCR of CD1, RD1-3

Image:PKU Switch 07-7-9 CD RD1 RD2 RD3.gif

l to r: CD1, RD1-3, Marker

Ligation of ECFP and pCC002 (C/N), SDD’ EGFP and pCC002 (B/C)

Retract lacZa^^ (S/X)

PCR of CD1, RD1 with temperature gradient

Temperature:

  • 1: 50.3C
  • 2: 53.2C
  • 3: 58.1C
  • 4: 63.5C
  • 5: 68.1C

Image:PKU Switch 07-7-9 RD1(gradient).gif

l to r: RD1(1-5), Marker

Image:PKU Switch 07-7-9 CD1(gradient).gif

l to r CD1(1-5), Marker

Retract RD1(4), CD1(5)

Ligation of CD1, RD1

Looking forward=

Transformation of RD1, CD1-pMD18T, ECFP-pCC002, SDD’-EGFP-pCC002

Precipitate pLX007 (X/S), SDD-EGFP (B/S) at 37C

Ligation of lacZa-pLX007 (X/S) and SDD-EGFP-pCC002(B/C)

检板子ECFP-pCC002

Temperature PCR of RD2, RD3, ligation, transformation

Personal tools