IGEM:Peking/2007/Switch-Notebook/2007-7-8

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Experimenter: LXL, NM, CCY

Contents

Contents

Send for sequencing RD1-1,2, SD1,2

Positive transformation of pLX007, 挑菌摇2个

pMDB与pMDB0的效率差别不大

(当然还是少了一点的)

看来感受态放在-40C还是可以的, 上次应该是运输的问题, 下次可以用liquid nitrogen直接带来

Colony PCR test of SD1,2, lacZa-pMD18T

  • primers: M13F/R
  • control: positive transformation of pMDB

Image:PKU Switch 07-7-8 SD1 1-12 SD2 1-10.gif

l to r: Marker, Negative control, SD1(1-12), Negative control, SD2(1-10)

Image:PKU Switch 07-7-8 SD2 11-12 lacZa 1-11.gif

l to r: Marker, SD2(11-12), Negative control, lacZa(1-11)

For sequencing tomorrow: SD1(1,4,5), SD2(1,2,3)

For miniprep tonight: lacZa(1,2,3)

ECFP^^失活, precipitate with enthanol

  • precipitate at 9:30am
  • collect at 2pm

Miniprep of ECFP1-5, mRFP1-20, 酶切检测

Image:PKU Switch 07-7-8 ECFP 1-5 mRFP 1-19.gif

l to r: ECFP(1-5), mRFP(1-19), Marker

  • ECFP is not ligated.
  • mRFP(2,4,14,18) are correct.

SDE-EGFP-pMD18T酶切

Image:PKU Switch 07-7-8 mRFP20 SDE-T1.gif

l to r: Marker, mRFP20, SDE-EGFP-pMD18T^^

What’s wrong?

Re-try:

Image:PKU Switch 07-7-8 SDE EGFP-T1 ECFP^^.gif

l to r: Marker, SDE-EGFP-pMD18T, ECFP^^

OK!

KOD PCR of SDD, SDD’_EGFP

  • Primers: SDD_EGFP_BamHI_F, SDD’_EGFP_BamHI_F (newly ordered) + GFP-ClaI-R
  • Template: SDD_EGFP_pMD18T

Image:PKU Switch 07-7-8 SDD SDD' EGFP KOD.gif

l to r: SDD, SDD', none, Marker, SDE-EGFP-pMD18T^^

切胶回收, 双切过夜 (BamHI & Cla I)

Result of pCC002-ECFP

Failed. pCC002自连太多, 于是失败. pCC002重切过夜.

ECFP^^回收, 连接, 过夜

  • ECFP, pCC002, ClaI/NotI

pCC002^^

Miniprep of pLX007, lacZa (xs)-pMD18T

No SalI.

Looking forward

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

Test of pLX007 and lacZa(xs)-pMD18T(1-3) by double digestion

pLX007(xs) 切载体, lacZa(xs)切片段, 回收? 连接?

ECFP-pCC002转化, 早上! 用Mach1-T1

pCC002^^ CN, SDD’_EGFP^^ BC失活, 沉淀, 回收

SDD-EGFP KOD PCR试温度梯度

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