Paul.xu: Created page with "1. After PCR digest Gateway cassette with enzymes 2. Ligate to vector of choice to generate destination vector. 3. Transform top10/DH5alpha, or STBL3, grow on LB/amp at 37 oC o..."

2012-03-29T14:00:01Z

Created page with "1. After PCR digest Gateway cassette with enzymes 2. Ligate to vector of choice to generate destination vector. 3. Transform top10/DH5alpha, or STBL3, grow on LB/amp at 37 oC o..."

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1. After PCR digest Gateway cassette with enzymes

2. Ligate to vector of choice to generate destination vector.

3. Transform top10/DH5alpha, or STBL3, grow on LB/amp at 37 oC overnight.

3. pick colonies, grow in 5 ml LB/amp.

1. Make miniprep, digest with BamH1 to see if chloramphenicol resistance gene is present (710 bp insert)

2. Usually, Top10 or STBL3 bacteria with gateway cassette will die when transformed with gateway cassette. But if competent cells is contaminated with ccdB Survival, the cell will grow.

Additional enzymes to cut: EcoR1 + Pst1 -->1125 bp if gateway cassette is present. <br>

[[Category:Protocols]]

Gateway-Vector Construction - Revision history

Paul.xu: Created page with "1. After PCR digest Gateway cassette with enzymes 2. Ligate to vector of choice to generate destination vector. 3. Transform top10/DH5alpha, or STBL3, grow on LB/amp at 37 oC o..."