IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Core/2009/07/10/Min Lin

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The assessment PCR last night has stoped.
PCR the SupD plasmid to standarlize this SupD part.
TIxi
Condition Gradient PCR, 50, 55, 60, 65 centigrade. And use both pfu and mastermix. Since mastermix is supposed to be easier to use.
Results.
All the temps works. Recycle the Pfu ones.

Cut the PCR recycle product with EcoRI&SpeI and also EcoRI and PstI
Cut B0015 terminator with EcoRI and XbaI.
Cut tetR plasmid with EcoRI and PstI to get tetR backbone.

16:26
Recycle SupD GEL, use 50ul EB to wash off.
The concentration of the recycle product is 35ng/ul
Use EcoRI and SpeI to cut SupD PCR product. Digestion starts from 18:40

19:40
And see whether the T7ptag and terminator vector has been digested.
CIP the terminator plasmid.
Recycle the backbone of tetR with GEL recycle Kit.

Recycle the T7ptag digestion product with DNA product recycle kit.

Ligation:

Ligase 1ul
Buffer 1ul
Terminator Vector 1.5ul
T7ptag Insert 6.5ul
Ligase 1ul
Buffer 1ul
tetR Backbone 1.5ul
T7ptag Insert 6.5ul
Ligase 1ul
Buffer 1ul
WSK digestion 1.2ul
SupD Insert 6.8ul