lab 208

Main purpose

1. Screening PCR on pZA21:RFP araBAD
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

Screening PCR on pZA21:araBAD:RFP spl

8 different conditions and 12 different annealing temperatures were screened.

• PCR mix according to standard protocol with adjusted amounts of water according to the amounts of DMSO and MgCl2 added to the reactions.

The conditions are:

Row A -> 1% DMSO + 0.04mM MgCl2 + GC buffer

Row B -> 3% DMSO + 0.04mM MgCl2 + GC buffer

Row C -> 5% DMSO + 0.04mM MgCl2 + GC buffer

Row D -> 5% DMSO + 0.5mM MgCl2 + GC buffer

Row E -> 5% DMSO + 2mM MgCl2 + HF buffer

Row F -> 3% DMSO + 1mM MgCl2 50mM + HF buffer

Row G -> 5% DMSO + 1mM MgCl2 50mM + HF buffer

Row H -> 5% DMSO + 0.04mM MgCl2 + HF buffer

• Primers - 51a and 51b1
• Template - "Ara" sample (plasmid isolation pZA21:araBAD:RFP).
• Annealing temperature: each condition were run in the following 12 different annealing temperatures:

1. 53.2 C
2. 53.5 C
3. 54.6 C
4. 56 C
5. 57.2 C
6. 58.4 C
7. 59.6 C
8. 60.6 C
9. 61.9 C
10. 63.2 C
11. 64.6 C
12. 65 C

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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