lab 208

Main purpose

Who was in the lab

Julia, Kristian, Henrike

Procedure

Colony PCR to verify inserts HAO and Nir in pZA21::ara and pZA21::ara tight

Colony PCR on the Nir transformants, testing for NirH (primers 45a, 45b, annealing 59C), NirM+S (primers 44, 11a annealing 64C) and NirN (primers 43, 11b annealing 68C).

program:

Colony PCR to verify HAO insert.

Primers: 28a, 28b

temperature: 59C

Results

Transfomation

Transformation on last friday gave the following results:

• HAO in pZA21::ara (from Biobrick K808000) - 6 colonies (3 orange and small, 3 normal color and very big)
• negative HAO in pZA21::ara (from Biobrick K808000) - no colonies

• HAO in pZA21::ara tight - a lot of colonies, some very big
• negative HAO in pZA21::ara tight - a lot of small colonies

• Nir in pZA21::ara tight - 8 colonies
• negative Nir in pZA21::ara tight - no colonies

Gel

Gel on the results of the first colony PCR on the Nir transformants, testing for NirH, NirM+S and NirN

• 1 kb ladder
• colony 1, NirH
• colony 1, Nir M+S
• colony 1, NirN
• colony 2, NirH
• colony 2, Nir M+S
• colony 2, NirN
• colony 3, NirH
• colony 3, Nir M+S
• colony 3, NirN
• colony 4, NirH
• colony 4, Nir M+S
• colony 4, NirN
• 1 kb ladder

The gel picture is inconclusive. We will do another colony PCR using the primers designed for sequencing.

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