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	[[Team:Tsinghua/Notebook/4_July_2011]]		[[Team:Tsinghua/Notebook/4_July_2011]]
-	Digest pET15b vector with NdeI + BamHI (Takara), 37 centigrade, 3 hours. (Approx. 1ug)	+	{{:Team:Tsinghua/Notebook/4_July_2011}}
-	Recycled digested vector, digested GFP fragment, digested OmpA fragment (previous day) via gel-extraction.	+
-	Ligation	+
-	1ul vector + 10ul GFP + 10ul Takara Solution I. RT, 1h	+
-	Transform DH5a competent cells, spread on Amp+ LB plates.	+
-		+
	[[Team:Tsinghua/Notebook/5_July_2011]]		[[Team:Tsinghua/Notebook/5_July_2011]]
-	Pick 6 colonies from the plates.	+	{{:Team:Tsinghua/Notebook/5_July_2011}}
-	Inoculate 5ml LB Amp+.	+
-	Culture PCR.	+
-	Dip less than 1 ul bacteria culture (~3hr in 37 centigrade) into a PCR reaction mix with GFP primers.	+
-	All of the clones have the GFP inserts	+
-	Mini-prep the vectors.	+

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Revision as of 14:19, 2 September 2011

Team:Tsinghua/Notebook/4_July_2011

pET15b OmpA-GFP construct

 Digest pET15b vector with NdeI + BamHI (Takara), 37 centigrade, 3 hours. (Approx. 1ug)
 Recycled digested vector, digested GFP fragment, digested OmpA fragment (previous day) via gel-extraction.
 Ligation
 1ul vector + 10ul GFP + 10ul Takara Solution I. RT, 1h
 Transform DH5a competent cells, spread on Amp+ LB plates.
 YX

pet15b OmpA-HIV cleavege site-sh3

 PCR OmpA-HIV site&HIV site-sh3
 overlapping pcr
 gel extraction

Team:Tsinghua/Notebook/5_July_2011

pET15b OmpA-GFP construct

 Pick 6 colonies from the plates.
 Inoculate 5ml LB Amp+.
 Culture PCR.
 Dip less than 1 ul bacteria culture (~3hr in 37 centigrade) into a PCR reaction mix with GFP primers.
 All of the clones have the GFP inserts
 Mini-prep the vectors.