Difference between revisions of "Team:UPO-Sevilla/Protocols"

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  Plasmid DNA extraction
 
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  DNA electrophoresis
 
  DNA electrophoresis

Revision as of 17:07, 4 October 2016

PROTOCOLS

Plasmid DNA extraction

  1. Collect the saturated bacteria culture in a 1'5 mL tube and centrifuge at 13000 rpm for 1 minute. Do it twice

  2. Add 100 μL of GTE (Tris HCl 25 mM, pH 8, glucose 50 mM, EDTA 10 mM), mix by inverting and incubate on ice for 5 minutes

  3. Add 150 μL of potassium acetate 3 M (pH 4'8), mix by inverting and incubate on ice for at least 5 minutes

  4. Centrifuge the tubes at 13000 rpm for 10 minutes and transfer the supernatant to a 1'5 mL tube

  5. Add 400 μL of ice ethanol 96% and centrifuge at 13000 rpm for 10 minutes

  6. Discard the supernatant, add 500 μL of ethanol 70% and centrifuge at 13000 rpm for 5 minutes

  7. Let the tubes dry and resuspend the plasmid DNA in 50 μL of TER (TrisHCl 10 mM, pH 8, EDTA 1 mM, RNAse 20 mg L -1)

DNA electrophoresis

GEL MAKING

  1. Prepare 40 or 100 mL of TAE (Trisacetate 40 mM, pH 7’7, EDTA 10 mM), it depends on the size of the gel

  2. Add 0’4 g or 1 g of agarose to the TAE solution (for 1% gel)

  3. Heat the solution until all the agarose has been dissolved and cold it to 50ºC

  4. Pour the the solution in the electrophoresis vessel, apply the combs and let it polymerize

GEL RUNNING

  1. Add 1 μL of loading buffer 6x (TrisHCl 5 mM, pH 8, EDTA 0’5 mM, glycerol 30%, xylene cyanol 2’5 g L -1, bromophenol blue 2’5 g L -1) to 5 μL of DNA sample

  2. Remove the combs from the gel, put the vessel on a horizontal electrophoresis system and cover it with TAE

  3. Pipette 6 μL of the DNA samples and ladder

  4. Run at 120 mV for 45 minutes approximately

REVEAL THE GEL

  1. Put the gel in ethidium bromide for 20 minutes

  2. Look the gel on the transilluminator and take a photo of it

Ligation

  1. Add 6 μL of insert DNA

  2. Add 1 μL of vector DNA

  3. Add 2 μL of ligase buffer high ATP (TrisHCl 50 mM, pH 7’6, MgCl 2 10 mM, DTT 5 mM, BSA 50 μg mL -1)

  4. Add 1 μL of T4 DNA ligase

  5. Incubate at 16ºC overnight

Generate competent cells