Lab 208

Main purpose

• PCR of AMO and HAO using USER primers and as a template using AMO and HAO extracted from chromosomal DNA with non-uracil primers
• PCR of Nir with new primers, template - cells of Pseudomonas
• verification of PCRs
• ON cultures and re-plating of Nir USER transformants from Colony PCR ( 17-07-2013)

Who was in the lab

Kristian, Gosia, Julia

Procedure

gel electrophoresis

ON cultures and re-plating of Nir USER transformants from Colony PCR

Positive Nir USER transformants obtained by colony PCR:

• Samples 25 and 26 were inoculated in 5 mL LB medium + 30 ug/ml kanamicyn for ON cultures preparation.
• Samples 25, 26 and 27 were re-plated in LB agar + 30 ug/ml kanamicyn

PCR to amplify AMO and HAO with USER primers

• AMO - primers 17a, 17b, 54 C annealing, 3 min of extension
• HAO - primers 18a, 18b, 59 C annealing, 3 min extension

Samples: 1,2 for AMO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 3 contains 5 uL of liquid culture of Nitrosomonas europea Samples 4,5 for HAO contain linear template - fragments extracted from chromosomal DNA with non-USER primers, sample 6 contains 5 uL of liquid culture of Nitrosomonas europea

PCR to amplify Nir

New primers which do not contain uracil, polymerase Phusion is used. Template is 5 uL of liquid culture of Pseudomonas.

Nir is being amplified in two fragments. Samples are as follows:

• 1.1 and 1.2 - First part of Nir, primers 41a, 41b, 63C annealing T, 9 min elongation
• 2.1 and 2.2 - Second part of Nir, primers 42a, 42b, 63C annealing T, 9 min elongation

Results

Gel 1

1. 1 kb ladder
2. AMO 5uL template, 1
3. AMO 5uL template, 2
4. AMO 10uL template, 1
5. AMO 10uL template, 2
6. HAO 5uL template, 1
7. HAO 5uL template, 2
8. HAO 10uL template, 1
9. HAO 10uL template, 2
10. restriction analysis cyc EcoRI
11. restriction analysis cyc HindIII
12. 1 kb ladder

Gel 2

1. 1kb ladder
2. pZA21 plasmid backbone, USER primers
3. pZA21 plasmid backbone, USER primers
4. Nir1, extraction PCR, 5uL template
5. Nir2, extraction PCR, 5uL template
6. Nir whole fragment, extraction PCR, 5uL template
7. Nir1, extraction PCR, 10uL template
8. Nir2, extraction PCR, 10uL template
9. Nir whole fragment, extraction PCR, 10uL template
10. Nir 1, 5 uL, U
11. Nir1, 10uL, U
12. Nir2, 5uL, U
13. Nir2, 10uL, U
14. Nir 25 colony PCR
15. Nir 26 colony PCR
16. Nir 27 colony PCR
17. Nir 28 colony PCR
18. 1kb ladder

Conclusion

The PCR of AMO and HAO with User endings was unsucessful. Restriction analysis of the plasmid containing cytochromes did not show the expected result.

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