IGEM:Peking/2007/Switch-Notebook/2007-7-10

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Experimenter: LXL, LCB

Contents

Contents

Precipitate with ethanol: SDD-EGFP^^, pLX007^^

  • Time: 8:30

Redo digestion of pLX007 with XhoI and SalI

Colony PCR test of ECFP-pCC002

  • Primers: pkem-F, pCC-test-R

Image:PKU Switch 07-7-10 ECFP-pCC002.gif

l to r: Marker, ECFP-pCC002(1-15), control

Select for incubation: ECFP-pCC002(6)

图像不清楚大约是因为Bobo错把TAE当了EB+TAE.

Transformation

  • SDD’-EGFP-pCC002
  • ECFP-pCC002
  • pCC002 (C/N)
  • CD1-pMD18T
  • RD1-pMD18T

Ligation

  • MCS-pCC009 4:1
  • pCC009 (self ligation control)

Retract of SDD-EGFP^^, pLX007^^

Sequencing result received

Temperature gradient PCR of RD2, RD3

  • Template: MG1655 FT Genome
  • Prime F: Rec_D_2_F/Rec_D_3_F
  • Prime R: Rec_S_2_R (new) / Rec_S_3_R (new)
  • Temperature: 50C, 53C, 58C, 63C, 68C

Image:PKU Switch 07-7-10 RD2.gif

l to r: RD2(1-5), ladder

不浓, RD3也不浓. 问什么我P的就不浓呢? 呼唤NM师兄!

Ligation

  • pLX007, lacZa 1:4
  • pLX007 (x/s), self ligation control
  • pCC002-SDD-EGFP

ExTaq PCR

  • 35 cycles
  • Product: SS1, SS2, SS3, SD2 (X/S), SD3
  • Template: Psul-pMD18T, Psul-pMD18T , Psul-pMD18T, SD2-pMD18T, SD2-pMD18T
  • Primer_F: Sul_S_1_F, Sul_S_2_F, Sul_S_3_F, lacO2_XhoI_F, Sul_D_3_F
  • Primer_R: Sul_S_1_R, Sul_S_2_R, Sul_S_3_R, lacO2_XhoI_R, Sul_D_3_R

Image:PKU Switch 07-7-10 SS1-3 SD2XS SD3.gif

l to r: SS1, SS2, SS3, SD2 (X/S), SD3, ladder

  • Retract
  • Ligate SS1-3, SD3, pMD18T

要是SD2 XS P不出来就惨了, 我还得重写引物, 明天温度梯度试试吧.

Transformation

  • 7 Amp+: SS1-T, SS2-T, SS3-T, SD3-T, CD1-T, RD1-T, pLX007 (xs)自, pLX007-lacZa
  • Cm+: SDD_EGFP_pCC002

Miniprep of ECFP-pCC002 C1-6

Looking forward

Colony PCR test of RD1, CD1, SS1, SS2, SS3, SD3-pMD18T

Colony PCR test of SDD’_EGFP_pCC002

SDD_EGFP_pCC002接着长过夜

Colony PCR test of lacZa-pLX007

Temperature PCR test of SD2 (X/S)

Enzyme digestion test of ECFP-pCC002 C1-6

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