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
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 
2. Analysis of Expression Level 
3. SDS 
4. Skim Milk Assay 
5. AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay 

Cloning

1. Enzymatic Digestion 
2. Dephosphorylation 
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 

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