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 [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 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 medium

For 1 L of LB medum:

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

Autoinduction medium

The Autoinduction medium is based on LB-medium. Add the following components after heat sterilization of 900 mL LB medium.

  • 5 mL of a 200 g L-1 steril L-rhamnose stock solution -> final concentration 2 g L-1
  • 2.5 mL of a 200 g L-1 steril glucose stock solution -> final concentration 1 g L-1
  • if necessary add antibiotics:
Cm: 1 mL or 3 mL of a 20 μg mL-1 Cm stock solution -> final concentration 20 or 60 mg L-1
Amp: 1 mL of a 100 μg mL-1 Amp stock solution -> final concentration 100 mg L-1
  • fill up to 1 L with steril ddH2O

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

SDS-PAGE gel

The following amouts are for one gel. Stacking gel 5 %:

  • 775 μL H2O
  • 1.25 mL 0,25 M Tris (pH 6,8)
  • 425 μL Bis/Acrylamide (0,8 %, 30 %)
  • 50 μL 5 % SDS
  • 25 μL 10 % Ammonium persulfate
  • 3 μL TEMED

Separating gel 12 %:

  • 1.5 mL H2O
  • 2.8 mL 1 M Tris (pH 8,8)
  • 3.0 mL Bis/ Acrylamide (0,8%, 30%)
  • 150 μL 5% SDS
  • 37.5 μL 10% Ammonium persulfate
  • 5 μL TEMED

SDS running buffer

  • 25 mM Tris [pH 8,3]
  • 192 mM Glycerol
  • 0.1 % SDS

4x Laemmli-buffer

  • 250 mM Tris-HCl
  • 40 % [v/v] Glycerol
  • 20 % [v/v] 2-Mercapthoethanol
  • 80 g L-1 SDS
  • 0.04 g L-1 BPB

Primers

This is a list of primers we have used.

primer name length sequence
F 28 GTTTCTTCGAATTCGCGGCCGCTTCTAG
R 29 GTTTCTTCCTGCAGCGGCCGCTACTAGTA
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 GTATCTCTCGAGAAAAGAGAGGCTGAAGCTACACGCAGGTGGTATGTATCACCTGCGTGTCTTGCTAGAT
TCTAATCAAG
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 44 ACGTGAATTCGCGGCCGCTTCTAGATGAACCTAGAAAAATTTGT
B.pumi_LAC_RV 41 CTGCAGCGGCCGCTACTAGTATTACTGGATGATATCCATCG
B.halo_FW 44 ACGTGAATTCGCGGCCGCTTCTAGATGAAAAAATCATATGGAGT
B.halo_RV 41 CTGCAGCGGCCGCTACTAGTATTACTCAGGCATATTTGGAA
T.thermo_LAC_FW 44 ACGTGAATTCGCGGCCGCTTCTAGATGCTGGCGCGCAGGAGCTT
T.thermo_LAC_RV 41 CTGCAGCGGCCGCTACTAGTACTAACCCACCTCGAGGACTC
E.coli_LAC_FW_T7 79 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCAACGTCGTGATTTCTT
E.coli_LAC_RV_HIS 62 CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTACCGTAAACCCTAACA
Xcc_LAC_FW_T7 79 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGTCATTCGATCCCTTGTC
Xcc_LAC_RV_HIS 62 CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTGCCTCCACCCGCACTT
B.pumi_LAC_FW_T779 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGAACCTAGAAAAATTTGT
B. pumi_LAC_RV_HIS 62 CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGCTGGATGATATCCATCG
E.coli_LAC_FW_T779 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCAACGTCGTGATTTCTT
E.coli_LAC_RV_HIS 62 CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGTACCGTAAACCCTAACA
T.thermo_LAC_FW_T7 79 ACGTGAATTCGCGGCCGCTTCTAGAGtaatacgactcactatagggagagaggagaaaaATGCTGGCGCGCAGGAGCTT
T.thermo_LAC_RV_HIS 62 CTGCAGCGGCCGCTACTAGTATTATTAGTGATGGTGATGGTGATGACCCACCTCGAGGACTC
Pc_lac35_FW_oS 38 acgtcacctgcgtgtagctgccatagggcctgtggcgg
Pc_lac35_RV 39 acgtcacctgcgtgtcaagTCAGAGGTCGCTGGGGTCAA
Tc_lac5_FW_oS 38 acgtcacctgcgtgtagctggtatcggtcctgtcgccg
Tc_lac5_RV 39 acgtcacctgcgtgtcaagTTACTGGTCGCTCGGGTCGC
Tv_lac5.P.FW 43 gaattcgcggccgcttctagATGTCGAGGTTTCACTCTCTTCT
Tv_lac5.S.RV 41 CTGCAGCGGCCGCTACTAGTATTACTGGTCGCTCGGGTCGC
Pc_lac35.P.FW 43 gaattcgcggccgcttctagATGTCGAGGTTCCAGTCCCTCTT
Pc_lac35.S.RV 41 CTGCAGCGGCCGCTACTAGTATCAGAGGTCGCTGGGGTCAA
J23117_RBS_FW 70 aattcgcggccgcttctagagttgacagctagctcagtcctagggattgtgctagcaaagaggagaaata
J23117_RBS_RV 70 ctagtatttctcctctttgctagcacaatccctaggactgagctagctgtcaactctagaagcggccgcg
J23103_RBS_FW 70 aattcgcggccgcttctagagctgatagctagctcagtcctagggattatgctagcaaagaggagaaata
J23103_RBS_RV 70 ctagtatttctcctctttGCTAGCATAATCCCTAGGACTGAGCTAGCTATCAGctctagaagcggccgcg
J23110_RBS_FW 70 aattcgcggccgcttctagagtttacggctagctcagtcctaggtacaatgctagcaaagaggagaaata
J23110_RBS_RV 70 ctagtatttctcctctttgctagcattgtacctaggactgagctagccgtaaactctagaagcggccgcg
J23103_K_FW 25 CTGACAGCTAGCTCAGTCCTAGGTA
J23110/117_K_FW 25 TTTACGGCTAGCTCAGTCCTAGGTA
T7_K_FW 26 TAATACGACTCACTATAGGGAAAGAG
CBDcex_2AS-Link_Frei 56 CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCGCCGACCGTGCAGGGCGTGC
CBDcex_Freiburg-Prefix 54 GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTCCGGCCGGGTGCCAGGTG
CBDcex_T7RBS 80 TGAATTCGCGGCCGCTTCTAGAGTAATACGACTCACTATAGGGAAAGAGGAGAAATAATGGGTCCGGCCGGGTGCCAGGT
CBDclos_2ASlink_compl 63 CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCTGCAAATCCAAATTCAACATATGTATC
CBDclos_Freiburg-Prefix 57 GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCTCATCAATGTCAGTTGAATTTTAC
Cex_Freiburg_compl 54 ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTGCCGACCGTGCAGGGCGTGC
Clos_Freiburg_compl 56 ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTGCAAATCCAAATTCAACATAT
Eco_Freiburg 53 ACGTGAATTCGCGGCCGCTTCTAGATGGCCGGCCAACGTCGTGATTTCTTAAA
Eco_Freiburg_compl 53 ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTACCGTAAACCCTAACATC
GFP_Freiburg 54 ACGTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC
GFP_Freiburg_compl 61 ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTTTGTATAGTTCATCCATGCCATGTGT
GFP_His6_compl 74 CTGCAGCGGCCGCTACTAGTATTAACCGGTGTGATGGTGATGGTGATGTTTGTATAGTTCATCCATGCCATGTG
GFP_FW 48 ATGCGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG
S3N10_Cex_compl 40 TTGTTGTTGTTCGAGCTCGAGCCGACCGTGCAGGGCGTGC
S3N10_Clos_compl 40 TTGTTGTTGTTCGAGCTCGAGCTGCCGCCGACCGTGCAGG
S3N10_GFP 40 CAATAACAATAACAACAACCGTAAAGGAGAAGAACTTTTC



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