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Lab 208

Main purpose

1.3.1 PCR for AMO 1.3.2 PCR for SPL (synthetic promoter library) 1.3.3 PCR for reference promoter 1.3.4 PCR for araBAD K808000 1.3.5 PCR to extract Nir2 from P. aeruginosa 1.3.6 PCR for araBAD and SPL with DMSO 1.3.7 PCR on Nir1 with MgCl2 gradient 1.3.8 Gel purification 1.3.9 Plasmid miniprep

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 (synthetic 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 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
• 2 - SPL, primers 52a, 52b1, template pZA21 with RFP, 50C, 2:30 min extension
• 3 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
• 4 - ref, primers 52a, 52b2, template pZA21 with RFP, 50C, 2:30 min extension
• 5 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
• 6 - araBAD, primers 12a, 12bn, template K80800, 50C, 2:30 min extension
• 1 kb ladder

AMO was gel purified.

Conclusion

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