lab 208

Main purpose

1. Screening PCR with Ara as template
2. PCR reaction in order to amplify backbone pZA21 containing araBAD and RFP
3. PCR reaction in order to amplify Nir1 and Nir2 fragments

Who was in the lab

Henrike, Kristian, Gosia, Julia

Procedure

PCR mix for backbone

• PCR mix - according to standard protocol.
• Primers - 1an and 1b
• Template - "Ara" sample (plasmid isolation pZA21:araBAD:RFP).
• Program was based on standard program with 65 C of annealing temperature and 4 min of extension time.
• Samples names: 1, 2, 3 and N (negative)

PCR for Nir1 and Nir2

• PCR mix according to standar protocol with changes: addition of DMSO in 3 different final concentrations (2%, 3%, 5%); two different buffers (HF 5x and GC 5x), amount of added water was dependent on volume of added DMSO.
• Primers for Nir1 - 39a, 39b
• Primers for Nir2 - 40a, 40b
• Templates - fragments Nir1 and Nir2 amplified with non-uracil primers, gel purified
• Polymerase x7
• Program (A99 - the same for Nir1 and Nir2) was based on standard PCR program with 50 C and 1 min of annealing parameters and 5 min of extension time.
• Samples names:

1. Nir1, GC buffer, 2% DMSO
2. Nir1, GC buffer, 3% DMSO
3. Nir1, GC buffer, 5% DMSO
4. Nir1, HF buffer, 2% DMSO
5. Nir1, HF buffer, 3% DMSO
6. Nir1, HF buffer, 5% DMSO
7. Nir2, GC buffer, 2% DMSO
8. Nir2, GC buffer, 3% DMSO
9. Nir2, GC buffer, 5% DMSO
10. Nir2, HF buffer, 2% DMSO
11. Nir2, HF buffer, 3% DMSO
12. Nir2, HF buffer, 5% DMSO

Results

Conclusion

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