Team:Aachen/Lab/Protocols

Protocols

General


  1. Polymerase Chain Reaction (PCR) 
    1. Colony-PCR using Alkaline PEG
    2. Colony-PCR with Heatshock
    3. Extended Colony-PCR
  2. Precipitation 
    1. test
  3. Cell Lysis 
    1. Lysozym
      1. Centrifuge the samples for 4 min at 4°C and 14000 rpm.
      2. Freeze the "dried" pellet overnight.
      3. Add 150 µL buffer on the pellet, resuspend it (e.g. with LB-medium) and incubate for 5 min at room temperature.
      4. Add 150 µL lysozym solution (c = 5-8 mg/mL).
      5. Incubate the samples for 1 h at 37°C and 250 rpm.
      6. Centrifuge the samples for 4 min at 4°C and 14000 rpm.
      7. Decant the supernatant.
    2. Sonication
      1. conditions:
      2. time:            3 min
      3. pulse on:    30 s
      4. pulse off:    20 s
      5. amplitude:  50%
    3. Glass beads
      1. Put some small glass beads into your sample tubes.
      2. Vortex your samples (5-10 min at level 2-6), avoid foam on the top of your sample.
      3. Put the used beads into a waste bottle filling with 70% EtOH for.

  4. Transformation 
    1. E. coli
      1. Thaw 100 µL E. coli DH5α or E. coli BL21(DE3) Gold on ice
      2. Add DNA (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; 1-4 µL PLICing-reaction) to the competent cells and swirl gently
      3. Incubate for 15-30 min on ice.
      4. Perform heatshock of the competent cells using a preheated water bath at 42°C for 45s (DH5α, BL21).
      5. Samples were immediately cooled down 5 min on ice.
      6. Fill up the transformation mix to 1 mL with SOC media and incubate at 37°C at 250rpm for 45 min (45-60 min) for recovery of the cells.
      7. Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench.
        1. 200 µL
        2. resuspended pellet (from centrifugation of the leftover)
      8. Incubate the agar plates at 37°C overnight(16-18 hours).
      9. Count single colonies of each agar plate on the next day.
    2. Sonication
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    3. Saccharomyces
  5. Preparation of Chemical Competent Cells  

Analytics


  1. Gelelectrophoresis 
      1. Carefully remove the comb of the prepared solid agarose gel.
      2. Put the gel into the gel-electrophoresis set up filled with buffer.
      3. Pipette 1 µL loading dye on a piece of parafilm.
      4. Add 5 µL of the sample to it and mix it by pipetting it up and down (avoid bubbles).
      5. Pipette the whole solution (6 µL), without air in the pipette tip, slowly into one well of the gel.
      6. Pipette 2.5 µL ladder into another well as a comparison (e.g. ladder 08: #SM0311).
      7. Let it run for 35 min: 300 mA, 90V, 50W.
      8. Take out the gel, analyze it and then discard the gel afterwards.
    2. Analysis of Expression Level 
      1. Preparation of pre-culture
        1. Fill 20 mL LB media into a (small) flask, add the corresponding antibiotics.
        2. Inoculate the pre-culture.
        3. Incubate your culture at 37°C, 250 rpm for 16-18 hours.
      2. Preparation of main culture
        1. Fill 200 mL LB media into a 1000 mL flask and add the corresponding antibiotics.
        2. Inoculate the main culture with 2-5 mL pre-culture, as to reach an OD600 of 0.1 in the main culture.
        3. Incubate your main culture at 37°C, 250 rpm till it reaches the OD600 of 1 (2-4 hours).
        4. Take a sample (500µL) out of your main culture and store it at -20°C.
        5. Induce the expression of your main culture by adding a final concentration of each 0.5 / 1 / 1.5 mM IPTG.
        6. Incubate at 37°C, 250 rpm for an hour.
        7. Take a sample hourly afterwards. (as described above)
      3. Analysis
        1. Perform a cell lysis with the samples which were taken and frozen at -20°C.
        2. Perform a Skim Milk Assay with every lysed sample.
        3. Perform a SDS-gel with all lysed samples.

    3. SDS 
    4. Skim Milk Assay 
    5. AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay 

    Cloning


    1. Enzymatic Digestion 
    2. Dephosphorylation 
      1. Dephosphorylation after restriction
        1. Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) after restriction (example: 1 µL for 2000 kb) and according buffer (volume according to concentration of buffer).
        2. Incubate at 37°C for 30–60 minutes.
        3. Stop reaction by heat-inactivation of rSAP at 65°C for 5 minutes.
        4. Store at -20°C.
    3. Ligation 
    4. Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM)  

    Media


    1. LB Medium 
    2. SC Minimal Medium 
    3. SOC Medium 
    4. M9 Medium 
    5. YEP Medium 

    Devices

    Kits

    1. Plasmid Isolation 
    2. PCR Clean-up 
    3. DNA Extraction from Agarose Gel 
    4. JET Cloning 


Link to our website
Wiki 2014 Aachen
Wiki 2015 Aachen
igem@rwth-aachen.de