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Team:Tsinghua/Notebook/16 August 2010
From 2010.igem.org
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== Module I, group 2(b) == | == Module I, group 2(b) == | ||
Universal primers of pUC19 are available now (marked p-up and p-do)! Use the universal primers to detect PEM, PKC and PEMKC with SuperMix system shown before. | Universal primers of pUC19 are available now (marked p-up and p-do)! Use the universal primers to detect PEM, PKC and PEMKC with SuperMix system shown before. | ||
| Line 6: | Line 5: | ||
The result shows that PEM plasmids are truly positive but kan gene were not ligated successfully. So that the EMKC are not positive with K missed. | The result shows that PEM plasmids are truly positive but kan gene were not ligated successfully. So that the EMKC are not positive with K missed. | ||
| + | == Module I, group 2c == | ||
| + | Run the same PCR program as yesterday and purify the product with gel purification kit. | ||
| + | ---- | ||
| + | |||
| + | Digest eGFP with XbaI and EcoRI, Ap is uesd to phosphorylate the 5' end of digestion result. | ||
| + | digestion system | ||
| + | eGFP 4ul | ||
| + | FD Buffer 2ul | ||
| + | XbaI 0.5ul | ||
| + | Dra III 0.5ul | ||
| + | AP 0.5ul | ||
| + | H2O 12.5ul | ||
| + | ---- | ||
| + | Digest mCherry fragment with Dra III,Ap is uesd to phosphorylate the 5' end of digestion result. | ||
| + | digestion system | ||
| + | mCherry 4ul | ||
| + | FD Buffer 2ul | ||
| + | Dra III 0.5ul | ||
| + | AP 0.5ul | ||
| + | H2O 13ul | ||
| + | ---- | ||
| + | Digest Kan fragment with Dra III and SpeI,Ap is uesd to phosphorylate the 5' end of digestion result. | ||
| + | digestion system | ||
| + | Kan 4ul | ||
| + | FD Buffer 2ul | ||
| + | Dra III 0.5ul | ||
| + | SpeI 0.5 | ||
| + | AP 0.5ul | ||
| + | H2O 12.5ul | ||
| + | ---- | ||
| + | Digest plasmid PSB1C13 with XbaI and SpeI, AP is used to phosphorylate the 5' end of digestion result. | ||
| + | digestion system | ||
| + | PSB1C13 4ul | ||
| + | FD Buffer 2ul | ||
| + | XbaI 0.5ul | ||
| + | SpeI 0.5ul | ||
| + | AP 0.5ul | ||
| + | H2O 12.5ul | ||
| + | ---- | ||
| + | |||
| + | 12h after digestion, the products are purified with kit. | ||
Latest revision as of 19:43, 26 October 2010
Module I, group 2(b)
Universal primers of pUC19 are available now (marked p-up and p-do)! Use the universal primers to detect PEM, PKC and PEMKC with SuperMix system shown before.
result
The result shows that PEM plasmids are truly positive but kan gene were not ligated successfully. So that the EMKC are not positive with K missed.
Module I, group 2c
Run the same PCR program as yesterday and purify the product with gel purification kit.
Digest eGFP with XbaI and EcoRI, Ap is uesd to phosphorylate the 5' end of digestion result. digestion system
eGFP 4ul FD Buffer 2ul XbaI 0.5ul Dra III 0.5ul AP 0.5ul H2O 12.5ul
Digest mCherry fragment with Dra III,Ap is uesd to phosphorylate the 5' end of digestion result. digestion system
mCherry 4ul FD Buffer 2ul Dra III 0.5ul AP 0.5ul H2O 13ul
Digest Kan fragment with Dra III and SpeI,Ap is uesd to phosphorylate the 5' end of digestion result. digestion system
Kan 4ul FD Buffer 2ul Dra III 0.5ul SpeI 0.5 AP 0.5ul H2O 12.5ul
Digest plasmid PSB1C13 with XbaI and SpeI, AP is used to phosphorylate the 5' end of digestion result. digestion system
PSB1C13 4ul FD Buffer 2ul XbaI 0.5ul SpeI 0.5ul AP 0.5ul H2O 12.5ul
12h after digestion, the products are purified with kit.
