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Lab 208

Main purpose

Make PCR on Nir with genomic DNA template. Amplify the pZE21 backbone with USER-primers for construction of the arabinose inducible system. Put His-tag on the cycAX construct.

Who was in the lab

Gosia, Kristian, Henrike, Julia

Procedure

PCR

• pZE21::GFP SF and pZE21::RFP in duplicates with primers pair 13 on 55C and extension time 3:00.
• cycAX in triplicates with primer pair 33 2 samples on 62C and 3:30 extension and 1 sample on 55C and extension 3:00.
• None negative was made for each containing same things as real samples just MQ instead of template DNA.

PCR of Nir on genomic DNA

Two kinds of templates were used for this PCR:

• isolated genomic DNA from P. aeruginosa
• isolated genomic DNA from P. aeruginosa digested with HindIII

names of samples:

• 1,2 - primers 41a, 41b - undigested genomic DNA - part 1 of Nir
• 3,4 - primers 42a, 42b - undigested genomic DNA - part 2 of Nir
• 5,6 - primers 41a, 41b - digested genomic DNA - part 1 of Nir
• 7,8 - primers 42a, 42b - digested genomic DNA - part 2 of Nir
• 9 - negative, primers 41a, 41b
• 10 - negative, primers 42a, 42b

Miniprep

Isolated plasmids from yesterdays ON cultures of AMO colony 2 and colony 3 using ethanol precipitation.

Gel purification

The two successful PCR products of the plasmid pZE21 (containing GFP SF, respective RFP) without promoter were subjected to DpnI treatment and gel purified with the QIAEXII gel extraction kit (no spin columns).

Results

Gel on PCR from yesterday

• 1kb ladder
• Nir 1uL genomic template
• Nir 3uL genomic template
• Nir 7uL....
• Nir 10uL....
• Nir 12uL....
• Nir 15uL....
• Nir 1uL HindIII treated genomic template
• Nir 3uL HindIII treated genomic template
• Nir 7uL....
• Nir 10uL....
• Nir 12uL....
• Nir 15uL....
• Negative control
• 1kb ladder

Gel on todays PCR

• 1 kb ladder
• GFP SF in pZE21 without promoter
• GFP SF in pZE21 without promoter
• RFP in pZE21 without promoter
• RFP in pZE21 without promoter
• negative control for no promoter (master mix without template)
• cycAX with His-tag at 55C
• cycAX with His-tag at 62C
• cycAX with His-tag at 62C
• neg. control for cycAX

Gel of Restriction analysis

• 1 kb ladder
• AMO colony 2
• AMO colony 3
• 1 kb ladder

Conclusion: The restriction analysis failed. We will load the uncut plasmid in differing concentrations on a gel to see if we can see a band.

Gel of Nir PCR

Gel of the PRC products from the Nir PCR today. See sample numbers in the entry in the procedure part.

• 1 kb ladder
• sample 1
• sample 2
• sample 3
• sample 4
• sample 5
• sample 6
• sample 7
• sample 8
• sample 9
• sample 10
• AMO in pZA21 from purification today (colony 2)
• AMO in pZA21 from purification today (colony 3)
• 1 kb ladder

Conclusion

No right bands where acquired from PCR-amplification with genomic DNA template.

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