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 with petri dish with transformed colonies instead of template plsmid DNA

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 tube

• Mix by pipetting up and down or flicking the reactions

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

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 30 or 50 μL EB buffer or water. Let stand for 1 minute and centrifuge for 1 minute

Phusion PCR

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