Agarose Gel

Materials

• 1X TAE

• Agarose

• Gel Red

• DNA samples

• 6X loading dye

• Nuclease free water

Procedure

Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments

• Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose

• Melt in microwave until agarose has melted (about 50 seconds)

• Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red

• Pour solution into agarose gel mold with comb

• Let set for 20 minutes or until solid

• Place gel in 1X TAE and remove comb

• Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease free water up to 12 μL

• Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)

• Take a picture of the gel at the UV detector

Amino acid solution

Materials

• Histidine-Hcl

• Uracil

• Leucine

• Tryptophan

Procedure

• Filter and sterilize solutions

• Add 8 mL per liter of selective medium or spread 500 μL on a selective plate

Colony PCR

Materials

• Materials for Taq PCR (except template plasmid DNA)

• Petri dish with transformed colonies

Procedure

• Prepare 25 μL reactions as in Taq PCR Protocol without template DNA

• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tubes

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol

Competent Cell Preparation – based on Open Wet Ware Protocol

Materials

• Bacterial overnight liquid culture

• Lysogeny broth (LB)

• CaCl2 solution, ice cold: 60 mM CaCl2, 15% glycerol, 10 mM PIPES, pH 7, filter sterilize and store at room temperature

Procedure

• Subculture overnight culture 1:100 in LB

• Incubate at 37°C with shaking until culture reaches an OD600 of 0.375

• Aliquot 20 mL if the culture into chilled 50 mL tubes

• Leave tubes on ice for 5 – 10 minutes

• Centrifuge cells at 1600 g for 7 minutes at 4°C

• Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution

• Centrifuge cells at 1100 g for 5 minutes at 4°C

• Discard supernatant and resuspend pellet in 4 mL ice cold CaCl2 solution

• Keep on ice for 30 minutes

• Centrifuge cells at 1100 g for 5 minutes at 4°C

• Discard supernatant and resuspend pellet in 800 μL ice cold CaCl2 solution

• Aliquot 100 μL of this suspension into microcentrifuge tubes

• Freeze in liquid nitrogen and store at -80°C

Gibson Assembly - based on NEB Gibson Assembly Protocol

Materials

• DNA fragments

• 2X Gibson Assembly Mater Mix (NEB)

• 2X NEBuilder Positive Control (NEB)

• Deionized water

Procedure

• Set up following reactions on ice, adding Gibson Assembly Master Mix last:

Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts

• Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)

• Store samples on ice or at -20°C until transformation

• Transform competent cells following the Transformation Protocol

Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)

Materials

• Bacterial overnight liquid cultures (1 - 5 mL)

• QIAprep Spin Miniprep Kit

Procedure

• Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes

• Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube

• Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times

• Add 350 μL N3 buffer and mix by inverting tube 4- 6 times

• Centrifuge for 10 min at 13000 rpm

• Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through

• Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through

• Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through

• Centrifuge for 1 minute to remove residual wash buffer

• Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute

Phusion PCR – based on NEB Phusion PCR Protocol

Materials

• 5X Phusion HF or GC Buffer

• dNTPs

• Forward and Reverse Primers

• Template plasmid DNA

• Phusion DNA polymerase

• Nuclease Free Water

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:

Usually 100 pg – 1 ng of template DNA is sufficient

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Guidelines

To be completed

Taq PCR – based on NEB Taq PCR Protocol

Materials

• 10X Standard Taq Reaction Buffer

• dNTPs

• Forward and Reverse Primers

• Template plasmid DNA

• Taq DNA polymerase

• Nuclease Free Water

Procedure

• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:

Usually 100 pg – 1 ng of template DNA is sufficient

• Mix by pipetting up and down or flicking the reactions

• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:

Guidelines

To be completed

PCR Product Purification - with QIAquick PCR Purification Kit (QIAGEN)

Materials

• PCR products

• QIAquick PCR Purification Kit

Procedure

• Add 5 volumes PB buffer to 1 volume of PCR product and mix

• Place QIAquick column in 2 ml collection tube

• Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through

• Wash by adding 0.75 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through

• Centrifuge QIAquick column for 1 minutes to remove residual wash buffer

• Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute

PEG/LiAc Solution

Materials

• 50% PEG (Polyethylene glycol) prepared with sterile deionized water

• 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved

• 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved

Procedure

• Prepare PEG/LiAc solution as follows:

Sd Medium<7h1>

Materials

• Amino Acid Powder

• Yeast Nitrogen Base

• Ammonium Sulphate

• Adenine Sulphate

• Water

• NaOH

• Agar

• Glucose

Procedure

• Place stirrer bar in 2 L Erlenmeyer

• Add 2.6 g amino acid powder, 3.4 g yeast nitrogen base, 10 g ammonium sulphate, 1 g adenine sulphate and 950 mL water

• Adjust pH to 5.9 by adding a few drops of 10 M NaOH

• In an other Erlenmeyer, add 35 g agar and 900 mL water

• Autoclave both bottles

• Transfer the content of first bottle to the agar-containing bottle

• Cool to 55°C

• Add 100 ml 40% glucose and 16 ml of the required amino acids

• Pour plates

Transformation – based on NEB Transformation Protocol

Materials

• Competent cells

• DNA

• SOC medium (SOB + Glucose)

• Petri dish with appropriate antibiotic resistance

Procedure

• Thaw competent cells on ice

• Add 2 μL DNA to the competent cells, mix by pipetting up and down or flicking the tube 4 -5 times

• Place mixture on ice for 30 minutes

• Heat shock at 42°C for 30 seconds

• Transfer tubes to ice for 2 minutes

• Add 950 μL room-temperature SOC media

• Incubate at 37°C for 60 minutes with shaking

• Spread 100 μL cells onto selection plates (warm plates to 37°C prior to this step for increased efficiency)

• Incubate overnight at 37°C