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

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Experimenter: LXL, NM, CDZ

The Day of COLONY PCR!

Contents

Contents

Sequencing result of SDE_EGFP_pMD18T received

Only No. 1 is correct.

Where is the plasmid? Culture?? Experiment notes???

等候处理.

Colony PCR test of ECFP-pCC002, mRFP-pCC002 (分区)

Primer

  • ECFP: pKan_F, EGFP_EcoRV_R
  • mRFP: pKan_F, mRFP_NotI_R

Image:PKU Switch 07-7-7 ECFP-pCC002 1-12 27-30 mRFP-pCC002 13-20.gif

l to r: ECFP 1-12, 27-30, mRFP 13-20, Marker

Image:PKU Switch 07-7-7 mRFP-pCC002 21-26.gif

l to r: mRFP 21-26, Marker

For Mini-prep tomorrow: ECFP 9, mRFP 14, 15, 21, 25, 26

mRFP看大小应该差不多, ECFP比较悬.

我也没有ClaI_SDY_F(Cla前没有保护碱基)的引物, 也不可能从T上往下切. 按理说CFP与mRFP也不差什么, 确认一下片段浓度, 没有问题就重连, 有问题就重切好了.

Colony PCR test of SD1,2-pMD18T

SD1,2-pMD18T grew very little

Primer: MBF/R

Image:PKU Swtich 07-7-7 SD1 1-8 SD2 9-20 T SulAT RD1-2-12.gif

l to r: SD1(1-8), SD2(9-20), pMDB, pSul-pMD18T, RD1-2(12), ladder

For sequencing tomorrow: SD1(1,4), SD2(1,2,6)

共长20个, P出来5个 (没P出来的都是什么)

Colony PCR test of RD1-1,2-pMD18T

昨天分区的没作阴性没有跑

Primer: MBF/R

Image:PKU Switch 07-7-7 RD1-1 RD1-2.gif

l to r: pMBD, RD1-1(1-12), RD1-2(1-11), ladder

Only RD1-1(11) is OK. 连T的效率这么低, 好奇怪啊.

再检一批.

Image:PKU Switch 07-7-7 RD1-1 13-35.gif

l to r: RD1-1(13-35), pMDB, ladder

Image:PKU Switch 07-7-7 RD1-2 13-35.gif

l to r: ladder, RD1-2(13-31), pMDB, RD1-2(32-35)

For sequencing:

  • RD1-1(11, 14, 22)
  • RD1-2(21, 29, 34)

Colony PCR test of lacZa_pLX007

Primers: test-F/test-R

lacZa_pLX007长了7个, pLX007自连长了13个. 昨天转化的连T都只有10个左右, 转化有问题啊.

Image:PKU Switch 07-7-7 LacZa1-7 ECFP mRFP.gif

l to r: lacZa1-7, pLX007, ladder

没有, 果然!

lacZa_pLX007应该调整一下. pLX007阳转, 用kit提. lacZa连T. 从T上酶切!!

Ligation

  • SD1-pMD18T, 4.5uL/0.5uL
  • SD2-pMD18T, 4.5uL/0.5uL
  • lacZa(SulI/XhoI)-pMD18T, 4.5uL/0.5uL
  • ECFP-pLX007, 4uL/1uL
  • pLX007自连, 1uL

3:00连接, 7:30转化

  • +pMDB positive
  • +pMDB 0 positive (用的是一直放在-40C的感受态)
  • +pLX007 positive

Test of the concentration of ECFP and mRFP

Image:PKU Switch 07-7-7 ECFP mRFP.gif

l to r: ladder, ECFP, mRFP

ECFP果然比较浅, 且用完了

KOD PCR ECFP

  • Primer: SDY_XFP_Cla2_F, XFP_NotI_R
  • Template: SDY_ECFP-pMD18T

切胶回收

Image:PKU Switch 07-7-7 ECFP KOD.gif

l to r: ECFP, ladder

Image:PKU_Switch_07-7-7_ECFP-after-extract.gif

l to r: 另一管ECFP^^, ladder, ECFP收 (2uL)

2uL一点都看不到, 扔掉.

Double cleave of ECFP

  • ECFP KOD 11uL
  • H 10uL
  • BSA 10uL
  • Triton 10 uL
  • ClaI 2uL
  • NotI 2uL
  • H2O 55uL

Total 100uL, overnight

倒平板

  • Amp+ 12 pieces
  • Cm+ 12 pieces
  • Liquid LB- one bottle

Looking forward

收pMDB, pMDB0的板子

比较转化率

SD1,2, RD1-1,2摇的菌送测序

CFP-pCC002的C1-5, mRFP-pCC002的m1-20提质粒, 酶切检测

Colony PCR test of SD1,2-pMD18T

阳性送测序

Colony PCR test of lacZa(XhoI/SulI)-pMD18T

阳性挑菌, 提质粒, 切片段

pLX007挑菌, 提质粒

ECFP-pCC002的板子晚上检测?

ECFP^^失活, 沉淀, 连pCC002转化?

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