Team:Bielefeld-Germany/Protocols/Materials

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Contents

Materials

</center> This is where we are going to list all our materials, devices and equipment that we have used.

Devices

Tecan Infinite Microplate Reader

Screen of our setup of the Infinite Reader

For measuring the Laccase activity we detected the level of oxidized ABTS via optical density at 420nm. The device we were able to use was a Tecan Infinite Reader M200. The program setup was in some parts adapted to the needs of our probes (like duration of the measurement) and in some parts standardized.
Used setup for Laccase activity measurements: Temperature: 25°C; Orbital shaking before each measuring cycle (time depends on duration of each cycle); Number of flashes: 30





Media, buffer and other solutions

Ampicillin stock solution

  • Solubilize 100 mg mL-1 Ampicillin
  • Store at -20 °C


Chloramphenicol stock solution

  • Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
  • Store at -20 °C


TAE buffer

For 1 L of 50 x TAE buffer you need:

  • 242.48 g Tris
  • 41.02 g Sodiumacetate
  • 18.612 g EDTA
  • Adjust pH to 7.8 with acetic acid
  • Solve in dH2O

10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer).

Briton Robinson Buffer

  • 0,1 mM acetic acid
  • 0,1 mM boric acid
  • 0,1 mM phosphoric acid
  • adjust to pH 5 with sodium hydroxide

DNA loading buffer

  • 50 % (v/v) glycerol
  • 1 mM EDTA
  • 0.1 % (w/v) bromphenol blue
  • Solve in ddH2O


LB media

For 1 L of LB media:

  • 10 g Trypton
  • 5 g Yeast extract
  • 10 g NaCl
  • 12 g Agar-Agar (for plates)
  • Adjust pH to 7.4


YPD media

For 1 L of YPD media:

  • 20 g Peptone
  • 10 g Yeast extract
  • 20 g Dextrose (add 50 mL sterile stock solution (40% dextrose))
  • Adjust pH to 6.5


Primers

This is a list of primers we have used.

primer name length sequence
pSB1C3-5aox1-f 60 CGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGAGATCTAACATCCAAAGACG
pSB1C3-5aox1-r 30 GGTGGCGGCGGGCGTTTCGAATAATTAGTT
5aox1-mfalpha1-f 68 AGAAGATCAAAAAACAACTAATTATTCGAAACGCCCGCCGCCACCATGAGATTTCCTTCAATTTTTAC
5aox1-mfalpha1-r 20 AGCTTCAGCCTCTCTTTTCT
mfalpha1-aarI-taox1-f 80 GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACACGCAGGTGGTATGTATCACCTGCGTGTCTTGCTAGATTCTAATCAAG
mfalpha1-aarI-taox1-r 20 TAAGCTTGCACAAACGAACT
taox1-phis4-f 60 GTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCATGCCATGGACAAGATTC
taox1-phis4-r 20 GGCCGCTCGAGTATTCAGAA
phis4-kozak-his4-f 72 AATAGTTTACAAAATTTTTTTTCTGAATACTCGAGCGGCCCCCGCCGCCACCATGACATTTCCCTTGCTACC
phis4-kozak-his4-r 30 TTATTATTTCTCCATACGAACCTTAACAGC
his4-3aox1-f 60 TCACCGCAATGCTGTTAAGGTTCGTATGGAGAAATAATAACGAGTATCTATGATTGGAAG
his4-3aox1-r 20 AAAACAAGATAGTGCCCCTC
3aox1-pSB1C3-f 60 AGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTTTTTACTAGTAGCGGCCGCTGCA
3aox1-pSB1C3-r 20 CTCTAGAAGCGGCCGCGAAT
taox-his4-f 61 GTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTAAGATCTCCTGATGACTGACTC
taox-his4-r 27 CTCGGATCTATCGAATCTAAATGTAAG
his4-3aox1-f02 60 TTATTTAGAGATTTTAACTTACATTTAGATTCGATAGATCCGAGTATCTATGATTGGAAG
his4_gi537483_f 46 ACGTgaattcgcggccgcttctagagAGATCTCCTGATGACTGACT
his4_gi537483_r 41 ctgcagcggccgctactagtaGATCTATCGAATCTAAATGT
B.pumi_LAC_FW ACGTGAATTCGCGGCCGCTTCTAGATGAACCTAGAAAAATTTGT
B.pumi_LAC_RV CTGCAGCGGCCGCTACTAGTATTACTGGATGATATCCATCG
Xcc_LAC_FW_T7 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGTCATTCGATCCCTTGTC
Xcc_LAC_RV_HIS CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTGCCTCCACCCGCACTT
E.coli_LAC_FW_T7 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCAACGTCGTGATTTCTT
E.coli_LAC_RV_HIS CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTACCGTAAACCCTAACA
T.thermo_LAC_FW_T7 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCTGGCGCGCAGGAGCTT
T.thermo_LAC_RV_HIS CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGACCCACCTCGAGGACTC


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