lab 208

Main purpose

Who was in the lab

Kristian, Henrike, Gosia

Procedure

PCR (promoter library)

PCR to amplify USER fragments of the constitutive SPL, the constitutive reference promoter and the arabinose reference promoter.

Used 5% DMSO, 2 uL of mM MgCl2 and GC-buffer.

constitutive SPL

• template: pZA21::RFP
• primers: 52a, 52b1
• annealing temp: 58.1C
• elongation time: 3:00

constitutive reference

• template: pZA21::RFP
• primers: 52a, 52b2
• annealing temp: 58.1C
• elongation time: 3:00

Wanted to make negative for the two above reactions but accidentally put the template in the master mix.

arabinose reference

• template: pZA21:araBAD:RFP (labeled Ara)
• primers: 51a,51b1
• annealing temp: 60.3C
• elongation time: 4:00

program (used for all 3 reactions):

PCR of Nir2 with USER primers

• PCR mix according to standard 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 Nir2 - 40a, 40b
• Template - fragment Nir2 amplified with non-uracil primers, 3uL were used
• Polymerase x7
• Program A99, samles 1-6, was based on standard PCR program with 55 C and 1 min of annealing parameters and 5 min of extension time.
• Program A1, samples 7-12, PCR with initial annealing temperature of 59C falling down to 53C (rate 0.1 C/min). 5 min of extension time.

Gel purification

Gel purified araBAD SPL from yesterdays screening PCR

Results

Gel of SLP screening PCR products (yesterday)

Each gel is representing one row; from left to right and up to down is row A to H:

Gel of todays PCR reactions

• 1kb ladder
• Nir2 PCR - sample 1
• Nir2 PCR - sample 2
• Nir2 PCR - sample 3
• Nir2 PCR - sample 4
• Nir2 PCR - sample 5
• Nir2 PCR - sample 6
• Nir2 PCR - sample 7
• Nir2 PCR - sample 8
• Nir2 PCR - sample 9
• Nir2 PCR - sample 10
• Nir2 PCR - sample 11
• Nir2 PCR - sample 12
• constitutive SPL 1
• constitutive SPL 2 (duplicate)
• constitutive promoter reference 1
• constitutive promoter reference 2 (duplicate)
• constitutive promoter reference 3 (very small sample, there was almost nothing left in the master mix)
• 1kb ladder

Conclusion

SLP screening PCR

The PCR run with condition E (5% DMSO, 2mM MgCl2 with GC buffer) had the highest flexibility; it yields the expected product at different annealing temperatures: 58.4 C, 59.6, C 60.6 C, 61.9 C, 63.2 C, 64.6 C, 65 C.

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