PET15b OmpA-GFP construct
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Revision as of 14:24, 2 September 2011
Team:Tsinghua/Notebook/3_July_2011
Contents |
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