Protocols

General

Polymerase Chain Reaction (PCR)
1. Colony-PCR using Alkaline PEG [1]
2. Preparation of alkaline PEG reagent:
3. Execution of the Colony PCR:
4. Colony-PCR with Heatshock [2]
  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 [2]
6. Aim:find a colony which carries the correct insert in the vector.
Precipitation
1. TCA precipitation [3]
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
Cell Lysis [2]
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.
Transformation
1. in Eschericha coli [4]
  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 [5]
  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
Preparation of Chemical Competent Cells
1. in Eschericha coli [4]
2. Preparation of TFB1 and TFB2 solution;
  TFB1:
3. TFB2:
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. in Saccharomyces cerevisiae [5]
6. Material: 0.1 M LiOAc (should be filter sterilized by using 0.2mm filter).
7. Competent Cells:

Analytics

Gelelectrophoresis [2]
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.
Analysis of Expression Level [2]
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 (500 µL) 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.
SDS
1. An intact SDS PAGE electrophoresis system should include: a tank, lid with power cables, electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or 15-well), and glass plates (a thin glass plate and a thicker one).
  The SDS PAGE gel in a single electrophoresis run can be divided into stacking gel and separating gel.. Stacking gel is poured on top of the separating gel (after solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample (details shown below).
2. Preparing the Gel:
  1. Content of separation gel:
    H2O : 2.1 ml
    30% acrylamide mix : 1.5 ml
    1,5M Tris (ph 8.8) : 1.3 ml
    10% SDS : 0.05 ml
    10% ammonium persulfate : 0.05 ml
    TEMED : 0.002 ml
  2. Content Stacking gel
    H2O : 0.72 ml
    30% acrylamide mix : 0.13 ml
    1,0 M Tris (ph 6,8) : 0.13 ml
    10% SDS : 0.01
    10% ammonium persulfate: 0.01 ml TEMED : 0.001 ml
3. SDS Protocol: [6]
  1. Use one thin glass plate in front of a thicker one.
  2. Put these two glass plates on a rubber sealing in the casting frame (make sure that you close the casting frame).
  3. Proof the closeness with water.
  4. Pour out the water and clean the device by using a papel towel.
  5. After water proofing of the casting frame, the separation gel (12%) could be prepared in a separate small beaker according to the protocol above.
    Note: Ammonium Persulfate and TEMED should be added at last, because TEMED will solidify the solution very fast. And be noticed that TEMED smells very bad, so use it fast and close the bottle rapidly.
  6. After making the separation gel solution should be filled between the two glass plates until it reaches the mark of the casting frame.
  7. You can straighten the surface by adding a thin layer of isopropanol to your separation gel.
  8. Keep the rest of your solution in the small beaker for cheaking the solidification of your separation gel.
  9. After the separation gel is solidified, the isopropanol should be discarded. Then clean the glass plates with water and discard the warter afterwards.
  10. Prepare the stacking gel in a separate small beaker according to the protocol above.
    Note: Ammonium Persulfate and TEMED should be added at last, because TEMED will solidify the solution very fast. And be noticed that TEMED smells very bad, so use it fast and close the bottle rapidly.
  11. Put the comb into the stacking gel.
  12. Let the stacking gel solidifying by following the same procedure from the protocol above.
    Note: Make sure a complete gelation of the stacking gel and take out the comb.
4. Running Setup
  1. Take the glass plates out of the casting frame and set them in the cell buffer dam.
  2. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
5. Prepare the samples:
  1. Mix your samples with sample buffer (loading buffer).
  2. Heat them in boiling water for 5-10 min.
6. Loading Samples and Runing the Gel
  1. Load prepared samples slowly into wells and make sure not to overflow. Don't forget loading protein marker (ladder).
  2. Then cover the top and connect the anodes.
  3. Set an appropriate volt and run the electrophoresis when everything's done.
    Note: As for the total running time, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about 1 hour for a 120V voltage and a 12% separating gel. For a separating gel posessing higher percentage of acylamide, the time will be longer.
7. Preparing gel after running
  1. Take out the sealed glass plates carefully.
  2. Clean the casting frame and used devices.
  3. Put the used running buffer into the used running buffer beaker for further usage.
8. SDS gel staining-destaining procedure
  1. Fix the gel with destaining solution for 10 min.
  2. Staining the gel with staining solution for 10-15 min.
  3. Perform the first destaining with the used destanng solution to for 15 min, pour back to the used destaning solution afterwards.
  4. Perform the second destaining with the fresh destaining solution and destain for 20-30 min. Afterwards pour back to the used destaining solution bottle.
  5. Use storage solution to destain for 1-2h, then discard the solution. ( optional)
Skim Milk Assay [7]
2. Principle
3. Protease activity can be detected by the clearance of the skim milk. This assay is suitable for library screening in microtiterplates.
4. Preparation of solutions
  1. 0.1M Tris/HCl buffer
    1. For a solution with 500mL dissolve6.05g Tris in 300mL ddH2O
    2. 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 0.5g skim milk in a beaker
    2. Stir for 2 minutes and directly start the reaction.
    3. and add 25ml of 0.1M Tris/HCl buffer.
5. Procedure
  1. Load 5-10 µL of enzyme solution in a 96-well flat bottom microtiter plate. Also use blank reactions.
  2. Add 190-195 µL of substrate solution<.
  3. Incubate for 15-20min at 23°C MTP spectrophotometer.
  4. Absorbance is measured at 650nm every minute (without shaking).
  5. Clearance(650nm)=Blank(650nm)–Sample(650nm)

Cloning

Enzymatic Digestion
1. Single digestion / linearisation (10 µL 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 (20 µL 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.
Dephosphorylation [8]
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 2000bp) 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.
Cloning of BioBricks
1. 1. Preparation
    1. Amplify the insert and the backbone, either with a PCR or a transformation and check concentrations (backbone should have a concentration of 200 ng/µL or more, insert of 100 ng/µL or more).
    2. Prepare agar plates and SOC media.
  2. First single digest of backbone and insert.
    1. Mix:
      20 µL DNA template
      2 µL restriction enzyme 1
      2 µL buffer
    2. Incubate both for 1h at 37°C, a heatkill is not necessary.
  3. PCR clean-up with additional nanodrop
    1. Elute in the end with 20 µL buffer.
    2. A high concentration is needed for further steps.
  4. Second single digest
    1. Mix:
      20 µL DNA template
      2 µL restriction enzyme 2
      2 µL buffer
    2. Incubate both for 1h at 37°C, a heatkill is not necessary.
  5. PCR clean-up
    1. Elute in the end with 20 µL buffer.
    2. A high concentration is needed for further steps.
  6. Dephosphorylation of backbone
    1. Dephosporylation of backbone with rSAP (according to protocol “Dephosphorylation”)
  7. PCR clean-up with additional Nanodrop of dephosphorylated backbone
    1. Elute in the end with 20 µL buffer.
    2. A high concentration is needed for further steps.
  8. Ligation of dephosphorylated and purified backbone with insert
    1. Mix:
      2.5 µL T4 ligase buffer X µL backbone (In the end in this mix should have a mass ratio backbone to insert of 1:10)
      20 µL insert
      2 µL T4 ligase
    2. It is recommended to prepare a control (1) sample with the same composition but with water instead of the insert.
    3. Incubate the samples at room temperature for one hour.
    4. Perform a heatkill for 10min at 65°C.
  9. Transformation
    1. Transform the ligation mix according to protocol “Transformation”
    2. Recommended controls:
      Control 1: Transformation of dephosphorylated backbone to check if dephosphorylation worked
      Control 2: Transformation of another plasmid with same antibiotics resistance to check if transformation worked
      Control 3: Plate wild type organism to check if antibiotics are working

Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM) [9]

Step 1

Mastermix (50 µl reaction)
Set up the mastermix on ice in a 0.2ml tube for PCR according to the table below. Thaw all non-enzyme components at room temperature, mix them by short vortex and collect them by short centrifugation. It is recommended to add PfuS DNA polymerase last in order to prevent any primer degradation caused by the 3' --> 5' exonuclease activity. Polymerase may be diluted in 1x buffer just prior to use in order to reduce pipetting errors.
Tube 1 - Forward Primer

	Amount	Final

Plasmid template(sequencing or column grade plasmid preparation)	X µl = 10-15 ng, max 50 ng
PDeionized water with PCR quality	39.5 µl-X µl
10 x PfuS buffer	5 µl	final 1x
X Forward primer (5 µM) [1]	5 µl	final 400 nM
dNTP-mix (10 mM each)	1 µl	Final 0.2 mM each nucleotide
PfuS(2U/µl) [2]	0.5 µl	Final 0.02 U/ul

Tube 2 - Forward Primer

	Amount	Final

Plasmid template(sequencing or column grade plasmid preparation)	X µl = 10-15 ng, max 50 ng
PDeionized water with PCR quality	39.5 µl-X µl
10 x PfuS buffer	5 µl	final 1x
X Forward primer (5 µM) [1]	5 µl	final 400 nM
dNTP-mix (10 mM each)	1 µl	Final 0.2 mM each nucleotide
PfuS(2U/µl) [2]	0.5 µl	Final 0.02 U/ul

PCR Program 1
1. 3 cycles
2. tube control with 50 µl
3. heated lid to 105°C

	Step	Time	temperature	Cycles

ID	Initial Denaturation	30 sec	98°C	3 cycles
D	Denaturation	10 sec [3]	98°C
A	Annealing	15-30 sec	X°C(45-72°C) [4]	Included in the 3 cycles
E	Extension	30-50 sec/kb [5]	72°C	Included in the 3 cycles
S	Storage		8°C

Note:

Extension time can be increased up to 2 min/kb if difficult template.

Step 2
1. Proceed with step 2 immediately after step 1 without freezing in between.
2. Mix the equal amount of foward (tube1) and reverse (tube2) reaction.
3. Add 1U=0.5µl PfuS DAN polymerase per 50 µl mixture.
PCR Program 2
1. 15 cycles
2. tube control with 50 µl
3. 105°C heated lid

	Step	Time	temperature	Cycles

ID	Initial Denaturation	30 sec	98°C
D	Denaturation	10 sec [3]	98°C	15 cycles
A	Annealing	15-30 sec	X°C(45-72°C) [4]
E	Extension	30-50 sec/kb [5]	72°C
FE	Final Elongation [6]	5-10 min	72°C
S	Storage		8°C

Note: Extension time can be increased up to 2 min/kb and/or 20 cycles if difficult templates, 1µl of PfuS might be necessary.
Gel and Purification
1. Load all samples on a 0.8% TAE agarose gel: 100V, 300 mA for 35min.
2. Check for optimal annealing temperature and if enough PCR product is present.
3. Purify the PCR products with the PCR Purification Kit. [7]
Dpnl digest the PCR product
1. Add 5 µl NEB bufferand 1 µl DpnI to the 45 µl purified PCR product.
2. Incubate at least for 2h at 37°C. [8]
3. Purify the PCR products with the PCR Purification Kit and elute in 30-50µl NE buffer.
Note: Efficiency can be increased by purification of the PCR-product before Dpnl digestion, especially when Phusion from NEB was used.

Transformation

The purified PCR products will be transformed into competent cells. See the protocol Transformation.

Helpful Notes
Numbers in brackets and with green background.
1. Primers: Oligonucleotide primers are generally 20-40 nucleotides long and ideally have a GC-content of 40-60%. The final concentration of each in a PCR reaction may be 0.2-1mM while 0.4µM is recommended.
2. PfuS DNA polymerase concentration: recommended use of 1 U per 50 µl reaction. However, the optimal concentration of PfuS DNA polymerase may vary from 0.5-2 U per 50 µl reaction depending on ampicon length and difficulty. DO not exceed 2U/50 µl reaction, especially for amplicons longer than 5kb.
3. Keep the denaturation as short as possible. Usually 5-10 seconds at 98°C is enough for most templates. Note: The denaturation time and temperature may vary depending on the ramp rate and temperature control mode of the cycler.
4. The PfuS DNA polymerase has the ability to stabilize primer-template hybridization. As a basic rule for primers >20 nt, anneal for 10-30sec at a Tm +3°C of the lower primer. For primers <20 nt use an annealing temperature equal to the Tm of the lower primer. Calculate Tm for matching sequence with IDT Oligoanalyzer
5. Extension time depends on ampicon length and complexity. For low complexity DNA (plasmid, lambda or BAC DNA) use extension time of 30sec per 1kb. For high complexity the extension time can be extended to 2min per 1kb.
6. Final Extension is an optional step which usually does not influence the outcome and can be skipped by default.
7. PCR purification can be skipped, depending on the buffer which is used during PCR. Dpnl is not active in Phusion HF-buffer from NEB.
8. Incubation time can be increased up to overnight for completely digestion.

Media

LB Medium [4]
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.
SC-U/SD-U Minimal Medium [10]
1. liquid
  1. For 1000mL medium mix:
    1. 6.7g yeast nitrogen base (without amino acids)
    2. 0.95g amino acid mix
    3. uracil
    4. 0.005% amino acid mix (aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine)
  2. Dissolve this mix in 800mL ddH2O.
  3. Autoclave at 15 psi, 121° C for 20min.
  4. Cool to 50°C and add 100mL of filter-sterilized 20% glucose or 200ml of filter-sterilized 10% raffinose.
2. Note: If you are making induction medium, follow Steps 1–3 above except dissolve the reagents only in 800 ml of deionized water. Cool the medium to 50°C and add 100 ml of filter-sterilized 20% galactose and 100 ml of filter-sterilized 10% raffinose to the medium when you want to induce your cells.
3. solid
  1. For 1000mL medium mix:
    1. 6.7g yeast nitrogen base (without amino acids)
    2. 0.95g amino acid mix
    3. uracil
    4. 0.005% amino acid mix (aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine)
4. For SD-U:
  1. 63mg histidine
  2. 75mg tryptophan
  3. 250mg leucine
SOC Medium [4]
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.
M9 Medium [11]
1. liquid
2. Prepare seven stock solutions for mixing the M9 medium in the end.
3. solid
  1. For preparation of 1L M9 medium for plates prepare 500mL concentrated liquid M9 medium and warm it up to 60°C.
  2. Dissolve 15g agar in 500mL ddH2O and autoclave this mixture for 15min at 121°C.
  3. Add the autoclaved agar solution to the M9 concentrate.
  4. Add antibiotics.
  5. Pour plates under sterile conditions.
YPD Medium
1. liquid
  1. For 1000mL mix:
    1. 20g peptone (1%)
    2. 20g dextrose (2%)
    3. 10g yeast extract (0.5%)
    4. Fill up to 1000mL mark with ddH2O.
  2. Autoclave the mixture at 15psi, 121°C for 20min.
2. solid
  1. For 1000mL mix:
    1. 10g peptone (1%)
    2. 20g Glc (2%)
    3. 5g Yeast extract (0.5%)
    4. 20g Agar
    5. Fill up to 1000mL mark with ddH2O.
  2. Autoclave the mixture at 15psi, 121°C for 20min.
  3. When medium cooled down add according antibiotics and make plates.

Devices

BioLector, in-house construction based on the established BioLector setup
Conductivity meter
Flow Cytometer
Molecular Viewer
NanoDrop
Plate Reader Tecan M1000
Ultra Sonicator

Kits

Plasmid Isolation
1. 1. NucleoSpin® Plasmid (NoLid) kit from Macherey-Nagel, 12/2015, Rev. 09, protocol p. 16-17
PCR Clean-up
1. 1. NucleoSpin® Gel and PCR Clean-up kit from Macherey-Nagel, 01/2012, Rev. 02, protocol p. 18-19
DNA Extraction from Agarose Gel
1. 1. NucleoSpin® Gel and PCR Clean-up kit from Macherey-Nagel, 01/2012, Rev. 02, protocol p. 20-21
JET Cloning
1. 1. Thermo Scientific CloneJET PCR Cloning Kit, protocol p. 5

References

[1]"Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood" (Chomczynski and Rymaszewsk, BioTechniques 2006)
[2] Information we got during our work in the Schwaneberg and Blank Lab
[3] http://www.koko.gov.my/CocoaBioTech/Purification%20Precipitation3.html
[4] Sambrook et al. 1989
[5] Joska et al. 2014
[6] http://www.assay-protocol.com/molecular-biology/electrophoresis/denaturing-page
[7] 2.4.5 Skim milk detection system http://publications.rwth-aachen.de/record/229540/files/4772.pdf
[8] NEB protocol for CIP alkaline phosphatase
[9] Wang, W. Y. and Malcom, B.A.: Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using Quikchange^TM site-directed mutagenesis. Biotechniques, 1999. 26(4): p.680-682
[10] Thermofisher pYES2 instructions
[11] Arie Geerlof Helmholtz Center Munich

Team:Aachen/Lab/Protocols