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From 2011.igem.org

Team:Tsinghua/Notebook/3_July_2011

pET15b OmpA-GFP construct

 PCR GFP fragment flanked by NdeI + BamHI from peGFP-N1 vector.
 Digest with NdeI, BamHI (Takara) 5 hours.
 PCR OmpA 540bp fragment from BL21 genome.
 Digest with NcoI (NEB) 3 hours.
 -YX

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.

Team:Tsinghua/Notebook/6_July_2011

pET15b OmpA-GFP construct

 Digest GFP vectors with NcoI (NEB) + CIP (NEB) 37 centigrade, 3hrs.
 Recycle the vector via isopropanol precipitation.
 Ligation
 5ul vector + 2ul OmpA inserts + Takara solution I mix. RT 1h
 Transform DH5a with all the reaction mix, spread on LB plates + Ampicillin.
 YX

Team:Tsinghua/Notebook/7_July_2011

pET15b OmpA-GFP construct

 Colony PCR
 Tip a tiny bit of the colonies, dip some on a new LB plate and dive the others into PCR reaction mix.
 Chose 20 clonies, 13 are correct.
 Inoculate 5 of the correct ones in 5ml LB Amp+
 -YX

pet15b OmpA-HIV cleavege site-sh3

 Digest OmpA-HIV cleavege site-sh3 with BamHI&NdeI
 gel extraction

Team:Tsinghua/Notebook/8_July_2011

pET15b OmpA-GFP construct

 Mini-prep OmpA-GFP vectors. Sent for sequencing.
 -YX