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

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Revision as of 08:12, 9 July 2007 by Chen Daizhuo (talk | contribs) (New page: Experimenter: NM & LCB ==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...)
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Experimenter: NM & LCB

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)!

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

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

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

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

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

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

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