lab 208

Main purpose

Who was in the lab

Kristian, Gosia, Natalia, Henrike

Procedure

PCRs

PCR for AMO

Made four reactions using 1 uL respectively 10 uL of culture from the -20 or from the glycerol stock (-80) as template and adjusted volume of water accordingly.

Primers 10a, 10b.

PCR for SPL (synthtic promoter library)

template: pZa21 with RFP, primers: 52a, 52b1, temperature: 50C, time: 2:30

PCR for reference promoter

template: pZa21 with RFP, primers: 52a, 52b2, temperature: 50C, time: 2:30

PCR for araBAD K808000

template: K808000, primers: 12a, 12bn, temperature: 50C, time: 2:30

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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