Team:Tsinghua/Notebook/16 August 2010

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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.