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

Experimenter: LXL, NM, CDZ

The Day of COLONY PCR!

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

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

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

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

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.

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

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.

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

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

l to r: ECFP, ladder

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

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