General

1. Polymerase Chain Reaction (PCR) 
   1. Colony-PCR using Alkaline PEG
   2. Preparation of alkaline PEG reagent:
   1. Combine 60 g PEG 200 (Sigma-Aldrich or equivalent) with 0.93 mL 2 M KOH and 39 mL water. If desired, KOH can be substituted by NaOH in the reagent.
      Note: PEG 200 is measured by mass rather than volume because of the viscosity of the liquid.
   2. Adjust the pH to 13.3–13.5..
      Note: Due to storage, some batches of PEG 200 have an acidic, rather than neutral pH. In this case, add an additional amount of alkali to reach the target pH range.
   3. Execution of the Colony PCR:
   1. Pick a bit of cell material of colony with a pipette tip and put it in a PCR tube with 50 µL of the reagent. Keep the pipette tip there during incubation time.
   2. Lyse the samples by incubation them in the prepared PEG reagent for 3-15 min at room temperature.
   3. Use 1-5 µL of the lysates as template in a 20-50 µL PCR reaction.
      Note: The aliquot of the sample lysate should not exceed 10% of the final volume of a PCR mixture.
   4. Colony-PCR with Heatshock
      1. Pick colonies from the transformed plates.
      2. Streak one part on a masterplate.
      3. Suspend the other part in 10 µL water.
      4. Incubate the suspended sample at 90°C for 5-6 minutes.
      5. Perform a PCR with primers that bind to the insert and with 1-5 µL of the lysate as template in a 20-50 µL PCR reaction.
   5. Extended Colony-PCR
   6. Aim:find a colony which carries the correct insert in the vector.
   1. Pick colonies of your plates.
      
      1. Streak a part of the colony on a master plate.
      2. Suspend the other part in master sample. (20 µL of ddH2O)
   2. Repeat step 1 eight times → eight colonies on each master plate and in each master sample.
   3. Repeat step 2 four times (or more) → four (or more) master plates/master samples.
   4. incubate the master plates at 37°C.
   5. Heat the master samples at 90°C for 6 minutes.
   6. Perform a Colony PCR with the master samples and with primers that anneal to the correct insert.
   7. Perform an agarose-gelelectrophoresis with the results from the colony PCR.
   8. If any sample shows a band with the correct size, then use this sample for the next step.
   9. Perform a Colony PCR with all eight colonies (taken from the master plate) that were part of the positive tested master sample.
   10. Perform another agarose-gelelectrophoresis and analysis with these samples.
   11. If any sample shows a band with the correct size, then use this sample for further experiments.
       Note:This experiment allows you to test a huge amount of colonies at the same time, and then to identify the exact colony which carries the correct insert.
   
   
2. Precipitation 
   1. TCA precipitation with Yeast cells
   2. Solutions
      1. SDS sample buffer:
         2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT or 125 mM Tris
         20% (v/v) Glycerol
         0.001% (w/v) Bromophenol Blue
         4% (w/v) SDS
      2. 50% TCA: 50% (w/v) Trichloroacetic Acid
      3. NaOH/Mercaptoethanol Solution:
         1% (v/v) 2-Mercaptoethanol
         0.25 M NaOH
   1. Grow yeast cells to a cell density of approximately an Optical Density at 600 nm (OD600) of 1 (1 X 107 cells/ml).
   2. Centrifuge the 1.5 ml of cells at 5,000 rpm in a microcentrifuge for 5 min.
   3. Remove the supernatant and resuspend the cells in 0.25 ml of NaOH/Mercaptoethanol Solution.
   4. Incubate the cells for 10 min on ice.
   5. Add 0.16 ml of 50% TCA.
   6. Incubate for 10 min on ice.
   7. Centrifuge at full speed in a microcentrifuge for 10 min.
   8. Remove the supernatant and resuspend the pellet in 1 ml of ice-cold Acetone.
   9. Centrifuge at full speed in a microcentrifuge for 10 min.
   10. Remove the supernatant and allow the pellet to air dry. Resuspend the pellet in 100-200 μl of SDS Sample Buffer.
   11. Proceed with SDS-PAGE and Western Blotting.
   
   
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. 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 

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