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. in Saccharomyces cerevisiae
      1. Combine 0.5 - 1 µg each of desired fragments, bring to a total volume of 34uL with ddH2O (for plasmids, use 100 - 300ng of plasmid DNA)
      2. 2. Prepare transformation mixture (Can be scaled up and prepared as master mix):
         240 ul 50% PEG 3350
         36ul 1M LiAc
         50ul single stranged carrier DNA
      3. Centrifuge aliquot of competent cells at 16000xG for 15s.
      4. Resuspend cells in 34ul (Plasmid) DNA solution.
      5. Add transformation mixture and vortex gently.
      6. Incubate tubes at 30°C for 30 minutes.
      7. Invert tubes, then incubate for precisely 30 min at 42°C.
      8. Centrifuge cells again at 16000xg for 15s.
      9. Resuspend pellet in 100ul dd H2O, spread on selection medium
         Note:If antibiotic marker is used, then outgrowth is needed:
         1. Resuspend cells in 500ul YPD medium and incubate for precisely 30 min at 42°C.
         2. centrifuge cells at 16000 x G, 15s.
         3. resuspend cells on 100ul ddH2O, then spread on medium
   
   
5. Preparation of Chemical Competent Cells  
   1. E. coli
   2. Preparation of TFB1 and TFB2 solution;
      TFB1:
   1. mix the components together:
      30mM k-Acetat
      50mM MnCl2 X 3H2O
      100mM RbCl2
      10mM CaCl2 x 2H2O
      15% glycerin in H2O
      pH=6.8, adjusted with KOH/ HCl
   2. sterile filtrate it under the clean bench
   3. TFB2:
   1. mix the components together:
      10mM MOPS
      75mM CaCl2
      10mM RbCl2
      15% glycerin in ddH2O
      pH=6.8, adjusted with KOH/ HCl
   2. sterile filtrate it under the clean bench
   4. Making competent E. coli cells:
      1. Prepare the TFB1 and TFB2 solution.
      2. Inoculate 5 mL overnight culture in LB-media and incubate at 37°C, 250rpm for 14h.
      3. Inoculate the main culture of 200 mL LB-media with 1-2% of the preculture (OD600 =0.1 at the beginning) in a 1 L flask and incubate it at 37°C, 250rpm until the OD600 = 0.4.
      4. Cool down the culture on ice and centrifuge it at 4°C, 3000 rpm, for 10 min.
      5. All following steps were carried out on ice. The pellet is resuspended in 15 mL sterile filtered cooled TFB1 solution and incubated for 10 min. Resuspend by gently swirling the tube, avoid to pipette the solution up and down.
      6. Centrifuge cells at 4°C, 3000 rpm for 10 min.
      7. Resuspend the pellets in 2-8 mL sterile filtered cooled TFB2.
      8. Divide competent cells in 50 µL or 100 µL aliquots in precooled Eppis and store the aliquots at -80°C immediately after freezing in liquid nitrogen
   5. S.cerevisiae
   6. Material: 0.1 M LiOAc (should be filter sterilized by using 0.2mm filter).
   7. Competent Cells:
   1. Prepare yeast overnight culture in YPD media.
   2. Inoculate main culture (50ml) with prepared overnight culture with OD600=0.2.
   3. Incubate for 4-5 h, until OD600=1 is reached.
   4. Centrifuge the culture at 2500xg for 5 min.
   5. Wash with 25 mL sterile ddH2O.
   6. Resuspend pellet in 1 mL 0.1 M LiOAc and transfer into a 1.5 mL Eppi.
   7. Centrifuge your sample at 10,000xg for 15 s.
   8. Resuspend pellet in 400 µL 0.1 M LiOAc.
   9. Divide the resuspended samples into 50 µL aliquots, store them at 4°C.
      Note: Fresh made cells can be stored for up to 1 week.
   
   

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 
   1. Skim Milk Assay
      1. Principle
         1. Protease activity can be detected by the clearance of the skim milk This assay is suitable for library screening in microtiterplates.
      2. Preparation of solutions
         1. Buffer: 0.1M Tris/HCl buffer
            1. For a solution with 500mL dissolve
               1. 6.05g Tris
               2. in 300mL ddH2O
            2. and adjust the pH to 8.6 using 1M HCl.
            3. Fill up the mixture to a volume of 500mL with ddH2O.
         2. Substrate solution:2%skim milk
            1. Weigh
               1. 0.5g skim milk
            2. in a beaker and add
               1. 25ml of 0.1M Tris/HCl buffer.
            3. Stir for 2 minutes and directly start the reaction.
      3. Procedure
      1. Load 5-10µL of enzyme solution in a 96-well flat bottom microtiter plate. Also use blank reactions.
      2. Add 190-195µL of substrate solution.
      3. Incubate for 15-20min at 25°C MTP spectrophotometer.
      4. Absorbance is measured at 650nm every minute (without shaking).
      5. Clearance(650nm)=Blank(650nm)–Sample(650nm)
   
   
5. AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay 

Cloning

1. Enzymatic Digestion 
   1. Single digestion / linearisation (10µL sample)
      1. Mix:
         1. 2 µL plasmid DNA (~100 ng)
         2. 1 µL restriction enzyme (e.g. EcoRI)
         3. 1 µL buffer (10x) for enzyme (e.g. Cutsmart for EcoRI)
         4. 6 µL ddH2O
      2. Incubate it for minimum one hour at 37°C.
   2. Double digestion (20µL sample)
      1. Mix:
         1. 2 µL Enzyme buffer (19x) (e.g.CutSmart)
         2. 0.4 µL restriction enzyme 1
         3. 0.4 µL restriction enzyme 2
         4. 10 µL DNA (~200 ng)
         5. 7.2 µL ddH2O
      2. Incubate it for minimum one hour at 37°C.
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 
   1. Liquid
      1. For 1000mL of liquid LB medium mix:
         1. 10g Peptone/ Tryptone (1%)
         2. 10g NaCl (1%)
         3. 5g Yeast extract (0.5%)
         4. Fill up with ddH2O to 1000 mL.
      2. Autoclave/ the mixture.
      3. Add desired antibiotics (under sterile conditions) when bottle is slightly cooled down.
   2. Solid
      1. For 1000mL of solid LB medium mix:
         1. 10g Peptone/ Tryptone (1%)
         2. 10g NaCl (1%)
         3. 5g Yeast extract (0.5%)
         4. 20g Agar
         5. Fill up with ddH2O to 1000 mL.
      
      
   3. SC Minimal Medium 
   4. SOC Medium 
      1. liquid
         1. For 500mL solution mix:
            1. 10g Tryptone
            2. 1g NaCl
            3. 2.5g yeast extract
            4. 480mL ddH2O
         2. Autoclave the mixture at 15psi, 121°C for 20min.
         3. Prepare three stocks:
            1. 1.0165g MgCl2(x6H2O) in 5mL ddH2O
            2. 1.2324g MgSO4(x7H2O) in 5mL ddH2O
            3. 1.9817g glucose monohydrate in 10mL ddH2O
         4. Sterile filtrate the solutions under sterile conditions.
         5. Add the three solutions to first 480mL mix under clean conditions as soon as the autoclaved mix is cooled down.
         6. Aliquote the medium and store at -20°C.
      
      
   5. M9 Medium 
   6. YEP Medium 
   
   

   Devices

   Kits

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