lab 208

Main purpose

Who was in the lab

Kristian, Gosia, Natalia, Henrike

Procedure

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

PCR to extract Nir2 from P. aeruginosa

PCR for araBAD and SPL with DMSO

Template and primers as previous.

PCR on Nir1 with MgCl2 gradient

Used previous isolation from a gel purification to make PCR for amplification of the strand. Used 5% DMSO as additive. The PCR was done with primer pair 41 and with x7 polymerase at 55C annealing and 5:00 min extension. Also we used 20 sec as denaturing time instead of the normal 10. The magnesium gradient was as follow:

• 0
• 1uL MgCl2 2mM per 50uL reaction
• 5uL MgCl2 2mM per 50uL reaction
• 20uL MgCl2 2mM per 50uL reaction

Gel purification

of the Nir1 fragment extraced from P. aeruginosa. Nanodrop: 8ng/uL

Plasmid miniprep

HAO in pZA21 from USER cloning was purified for restriction analysis. Nanodrop measurement: 180ng/uL

Results

Gel 1

1% agarose gel

• 1 kb ladder
• Nir1 col. Taq
• Nir1 col. Taq
• Nir2 col. Taq
• Nir2 col. Taq
• Nir1 frag. Taq
• Nir1 frag. Taq
• Nir1 col. x7
• Nir1 col. x7
• neg
• Hi spec. buffer
• Hi spec. buffer
• DMSO
• DMSO
• Nir1
• Nir1
• Nir2
• Nir2
• 1 kb ladder

Nir1 was purified.

Gel 2

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

Gel 3

1% agarose gel

• 1 kb ladder
• AMO, 1uL of -20 culture as template
• AMO, 10uL of -20 culture as template
• AMO, 1uL of glycerol stock as template
• AMO, 10uL of glycerol stock as template
• neg from AMO PCR
• 1
• 2
• 3
• 4
• 5
• 6
• 1 kb ladder

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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