Lab 208

Main purpose

• Extraction PCR for AMO
• PCR for SPL (synthetic promoter library)
• PCR for reference promoter
• PCR for araBAD biobrick K808000
• PCR to extract Nir2 from P. aeruginosa
• PCR for araBAD and SPL with DMSO
• PCR on Nir1 with MgCl2 gradient
• Gel purification of Nir1 (from P. aeruginosa)
• Plasmid isolation: HAO in pZA21 from USER cloning.

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 extracted 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

• Nir was obtained from colony PCR and purified.
• AMO was extracted and gel purified.

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