Team:Shenzhen SFLS/Notebook

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<h3>Enzyme</h3>
<h3>Enzyme</h3>

Latest revision as of 04:02, 21 June 2014

Notebook

Material

Bacteria strains

Strain name Risk group
E.coli strain DH5α 1
E.coli strain Mg1655 1
E.coli strain BL21 1

Primer

Name Sequence Length
LGT F ctggaattcatcgatcgttctagagaggccgccgcaaaagaa 42
LGT R ggactgcagattattatactagtattaagctctagtggaaacctgt 46
SH3 F cgctggctggtttagttttagcgtttagcgcaagcgcagctgaggccgccgcaaaagaa 59
SH3 F2 ctggaattcgcggccgcttctagatgaaaaagatttggctggcgctggctggtttagt 58
SH3 R ggactgcagcggccgctactagtatacttctccacgtaagggac 44

Plasmid

Enzyme

name manufacturer
QuickCut™ EcoRI Takara
QuickCut™ SpeI Takara
QuickCut™ PstI Takara
QuickCut™ XbaI Takara
T4 DNA Ligase Takara
rTag PCR enzyme Takara
Q5® High-Fidelity 2X Master Mix New England Biolab

Marker

name manufacturer
200bp DNA ladder marker Takara
BlueRAY Prestained Protein Ladder Jet Biofil

Kit

name manufacturer
TIANprep Mini Plasmid Kit DP103 Tiangen
TIANgel Midi Purification Kit DP209 Tiangen
Aflatoxin B1 Elisa kit Reagen

Buffer

name manufacturer
10X QuickCut Green buffer Takara
10X QuickCut buffer Takara
10X T4 DNA ligase buffer
dNTP Mixture Takara
10X PCR buffer Takara

Competent Cell

name manufacturer
DH5α competent cell Tiangen
BL21 competent cell Tiangen

Protocol

Transformation

  1. Remove 30-50 μl competent cell into a clean 1.5 ml micro-centrifuge tube
  2. Add 100ng plasmids or ligation products to the competence
  3. Let stand on ice for 30 minutes
  4. Heat shock for 90 seconds at 42°C
  5. Let stand on ice for 5 minutes
  6. Add 500 μl LB into the micro-centrifuge tube and incubate at 37°C for 45minutes
  7. Centrifuge for 3 minutes at 3000rpm and leave 100 μl supernatant
  8. Blow the bacteria up and add the bacteria into the corresponding plate
  9. Incubate at 37°C overnight

Plasmids Purification

  1. Remove 2ml overnight cultures bacteria into a micro-centrifuge tube, centrifuge for 1 minutes at 12000rpm and discard the supernatant (repeat it for one time)
  2. Cell lysis
    1. Add 250 μl Buffer P1
    2. Add 250 μl Buffer P2 and gently flip top to bottom for 6~7 times
    3. Add 350 μl Buffer P3 and gently flip top to bottom for 6~7 times Centrifuge for 10 minutes at 13000rpm
  3. Wash silica membrane of the spin column with 500 μl Buffer BL , centrifuge for 1 minutes at 12000rpm and and discard the waste liquid
  4. Remove the supernatant (from 3) to the spin column and let stand for 2 minutes
  5. Centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  6. Add 600 μl Buffer PW , centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  7. Add 600 μl Buffer PW , centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  8. Centrifuge for 3 minutes at 13000rpm and discard the waste liquid
  9. Put the column into a clean 1.5 ml micro-centrifuge tube and dry silica membrane for 10 minutes
  10. Add 50 μl ddH2O (heated up to 55°C) and Add to the center of the column
  11. Centrifuge for 1 minutes at 12000rpm to collect eluted DNA

Restriction Enzyme Digestions

  1. Calculate the following requirement for enzymes reaction

    name amount
    10x Quickcut Buffer 5μl
    DNA solution 1μg DNA
    Enzyme(EcoRI or XbaI or SpeI or PstI) 1μl per kind
    ddH2O Add to 50μl
  2. Incubate at 37°C for 40 minutes

  3. Analyze by gel electrophoresis

Agarose Gel Electrophoresis

  1. Confect a mixture in concentration of 1.5 ng agarose per 100 ml 1xTAE
  2. Boil up the mixture until the agarose completely dissolved
  3. Cool down to 50°C to 60°C
  4. Pour it into a flat-bed tray and insert a comb
  5. Put into the running buffer (1x TAE buffer)
  6. Load the DNA mixture with loading buffer into the pockets
  7. Electrophoreses for 35 minutes at 120V

Gel Extraction Of DNA Fragments

  1. Get the gel parts containing the needed DNA fragement size from the agarose gel with a scalpel
  2. Centrifuge for 30 seconds at 12000rpm
  3. According to the volume of the gel and add Buffer PN of the same volume
  4. Incubate at 55°C until the gel slices completely dissolved
  5. Wash silica membrane of the spin column with 500 μl Buffer BL , centrifuge for 1 minutes at 12000rpm and and discard the waste liquid
  6. Remove the liquid (from 4) to the spin column
  7. Centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  8. Add 600 μl Buffer PW , centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  9. Add 600 μl Buffer PW , centrifuge for 1 minutes at 12000rpm and discard the waste liquid
  10. Centrifuge for 3 minutes at 13000rpm and discard the waste liquid
  11. Put the column into a clean 1.5 ml micro-centrifuge tube and dry silica membrane for 10 minutes
  12. Add 25 μl ddH2O (heated up to 55°C) and Add to the center of the column
  13. Centrifuge for 1 minutes at 12000rpm to collect eluted DNA

Ligation

  1. Calculate the following requirement for enzymes reaction

    name amount
    10x T4 Buffer 2μl
    large fragments 30ng DNA
    small fragments 60ng DNA
    T4 ligase 1μl
    ddH2O Add to 20μl

    The reaction solution was adjusted according to annealing conditions.

  2. Incubate at 16°C for 1 hour

Colony PCR

Colonies were picked with pipet tips and dipped into the PCR mixture.

ingredient amount
Overnight Culture Top of a pipet tip 30ng DNA
10x PCR buffer 2.0μl
Forward primer (10µM) 0.4μl
Reverse primer (10µM) 0.4μl
dNTPs mixture 2.0μl
Polymerase 0.2μl
ddH2O Add to 20μl

Storage:

name temperature [°C]
plasmids and engineered bacteria -20
competent cell -80
plates with bacteria 4
other chemicals As the protocol recommends

Schedule

  • Week1 making mistakes and improving the project

  • Week2 making mistakes and improving the project

  • Week3 making mistakes

  • Week4 take a break

  • Week5 gene synthesising

  • Week6 constructing the device

  • Week7 constructing the device

  • Week8 constructing the device

  • Week9 making mistakes

  • Week10 making mistakes

  • Week11 constucting the device

  • Week12 doing western blot

  • Week13 doing western blot

  • Week14 Elisa

  • Week15 writing wiki

  • Week16 go to U.S and precentation

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