Lab 208

Main purpose

Find out what is wrong with Nir-extraction and why we make a successful USER-reaction with any USER-products from HAO and AMO.

Who was in the lab

Gosia, Kristian, Henrike

Procedure

Gel casting and loading

1% agarose gels were prepared with 5% Ethidium bromide.

Results

Verification gel

1% gel to verify our earlier PCR purifications

wells (in bracets position of the purification in the gene box):

• 1: broad band ladder
• 2: HAO extracted from N. europaea with flanking primers (A4)
• 3: AMO extracted from N. europaea with flanking primers (A6)
• 4: cycAX extracted from N. europaea with flanking primers (A5)
• 5: Nir extracted from P. aeruginosa with flanking primers (A7)
• 6: Nir extracted from P. aeruginosa with flanking primers (A8)
• 7: Nir extracted from P. aeruginosa with flanking primers (B8)
• 8: Nir extracted from P. aeruginosa with flanking primers (B9)
• 9: pZA21 USER fragment, DpnI treated (F1)
• 10: cycAX USER fragment (G1)
• 11: AMO USER fragment (G2)
• 12: HAO USER fragment (G3)
• 13: Nir USER fragment (G4)
• 14: AMO USER fragment (G5)
• 15: 1kb plus ladder

colony PCR gel

1% gel to analyse yesterday's colony PCR

wells:

• 1: 1 kb ladder
• 2: HAO colony 1
• 3: HAO colony 2
• 4: RFP in pZA21 without promoter
• 5: RFP in pZA21 without promoter
• 6: AMO colony 3 (count starts from 3)
• 7: AMO colony 4
• 8: AMO colony 5
• 9: AMO colony 6
• 10: AMO colony 7
• 11: AMO colony 8
• 12: AMO colony 9
• 13: AMO colony 10
• break in the gel
• 14: cycAX colony 1
• 15: cycAX colony 2
• 14: RFP colony 1
• 14: RFP colony 2

Conclusions

We USER-procucts seems to be fine and we then speculate that the reason why we only see cycAX as being successful in the transformation is due the destroying effect of too many membrane proteins. We will try to solve this by putting HAO and AMO under an inducible promoter. The pZA21::RFP transformation seems to be successful on colony-PCR and this is also visual from the red colonies.

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