lab 208

Main purpose

Who was in the lab

Kristian, Gosia, Natalia, Henrike

Procedure

PCR

lots of PCR done

gel purification

of the Nir1 fragment extraced from P. aeruginosa

plasmid miniprep

HAO in pZA21 from USER cloning was purified for restriction analysis

Results

1% agarose gel

• 1 kb ladder
• Nir1 col. T
• Nir1 col. T
• Nir2 col. T
• Nir2 col. T
• Nir1 frag. T
• Nir1 frag. T
• Nir1 col. X7
• Nir1 col. X7
• neg
• Hi
• Hi
• DMSO
• DMSO
• Nir1
• Nir1
• Nir2
• Nir2
• 1 kb ladder

1% agarose gel

• 1 kb ladder
• NirW
• NirW
• araBAD biobrick K808000
• araBAD biobrick K808000
• SPL in pZA21 containing RFP
• SPL in pZA21 containing RFP
• reference promoter in pZA21 containing RFP
• reference promoter in pZA21 containing RFP
• neg
• neg
• 1 kb ladder

(pic small gel)

Gel result:

• 1 - ladder
• 2 - sample nr 1 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
• 3 - sample nr 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
• 4 - sample nr 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
• 5 - sample nr 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
• 6 - sample nr 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
• 7 - sample nr 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
• 8 - sample nr 7 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
• 9 - sample nr 8 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
• 10 - sample nr 9 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
• 11 - sample nr 10 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
• 12 - sample nr 11 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, Phusion polymerase
• 13 - sample nr 12 - - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, x7 polymerase
• 14 - sample nr 13 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
• 15 - sample nr 14 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
• 16 - sample nr 15 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
• 17 - sample nr 16 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
• 18 - ladder

Conclusion

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