Calgary/protocols

• Full Procedure
• Protocols and Techniques
• Parts and Primers
• Printer System
• Results

Bacterial Transformation

• Thaw 100 ul of competen cells (per transformation) on ice just before they are needed
• Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
• Heat shock for 2 minutes at 42 degrees Celsius or 5 minutes at 37 degrees Celsius
• Place on Ice for 5 minutes
• Add 250ul SOC medium to each tube
• Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmides always use one hour)
• Spin down to remove all supernatant except approximately 100 ul
• Plate approximately 30 ul on each of two antibiotic plates
• Grow overnight at 37 degrees Celsius

For this protocol we used three controls

• Positive Control - pBluescript in TOP10 cells on amp resistant plates
• Negative Control - TOP10 cells grown on amp resistant plates
• Negative Control - DB31 cells on amp resistant plates

Rehydration

Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used.

• Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want
• Add 15 ul of diH20 (deionized water)
• Take 1 ul DNA and transform into your desired competent cells, plate out onto a plate with the correct antibiotic and grow overnight. Your goal here is to obtain single colonies

For this protocol we used three controls

• Positive Control - pBluescript in TOP10 cells on amp resistant plates
• Negative Control - TOP10 cells grown on amp resistant plates
• Negative Control - DB31 cells on amp resistant plates

PCR Protocol

Thermocycler Conditions

• 1 Cycle - 6 minutes at 95 degrees Celsius
• 36 cycles
  • 1 minute at 95 degrees Celsius
  • 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content )
  • 1 minute at 72 degrees Celsius
• 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius

Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 to 3 minutes

Plasmid Preparation Protocol - from GenElute Plasmid Miniprep Kit

• Harvest Cells
  • pellet 1-5 ml of an overnight culture.
• Resuspend Cells
  • Completely resuspend the bacterial pellet with 200ul of resuspension solution. Vortex or pipette up and down to throroughly resuspend cells until homogenous. Incomplete suspensions will result in poor recovery.
  • Another rapid way to resuspend the cell pelles is to scrape teh bottoms of the microcentrifuge tubes back and forth five times across the surface of a polpropylene microcentrifuge tube storage rack with 5 X 16 holes
• Lyse Cells
  • Lyse resuspended cells by adding 200 ul of the lysis solution. Immediately mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous. Do Not Vortex .
• Neutralize
  • Precipitate the cell debris by adding 350 ul of the Neutralization/Binding solution. Gently invert the tube 4-6 times. Pellet the cell debris by centrifuging at maximum speed for 10 minutes.
• Prepare Column
  • Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. add 500 ul of the Column Preparation Solution to each miniprep column and centrifuge at Max speed for 30 to 60 seconds. Discard the flow through liquid.
• Load Cleared Lysate
  • Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at Max speed for 30 to 60 seconds. Discard the flow through
• Wash Column
  • Add 750 ul of the diluted Wash Solution to the column. Centrifuge at Max speed for 30 to 60 seconds. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional wash solution to remove excess alcohol
• Elute DNA
  • Transfer the column to a fresh collection tube. Add 100ul of Elution Solution or molecular biology reagent water to the column. For DNA sequencing and other enzymatic applications, use water or 5mM tris-HCL, pH 8.0, as an eluant. Centrifuge at Max speed for 1 minute. DNA is now present in the eluate and is ready for immediate use or storage at -20 degrees Celsius.

Construction Technique

Determine the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning.

In the Insert Tube...

• 600ng of DNA (To figure out the volume, the calculation is 600 / concentration of plasmid. This gives you volume in μL).
• Water, so that the volume of DNA and water in the tube is 35 μL
• 4 μL of React 1 Buffer
• 0.5 μL of EcoR1
• 0.5 μL of Spe1

In the vector Tube...

• 250ng of DNA (To figure out the volume, the calculation is 250 / concentration of plasmid. This gives you volume in μL).
• Water, so that the volume of DNA and water in the tube is 35 μL
• 4 μL of React 2 Buffer
• 0.5 μL of EcoR1
• 0.5 μL of Xba1

Put both tubes into the 37°C water bath for one hour. After, place them into the 65°C heating block for 10 minutes. This destroys any enzymes in the tube (which is ok, because by now they’ve done all they need to). Take the insert out, and put it in a -20°C freezer.

To the vector tube, add 5 μL of 10x Antarctic Phosphatase Buffer, 4 μL of water, and 1 μL of Antarctic Phosphatase. We do this to prevent the vector from closing up again without any insert. Put the tube into the 37°C water bath for 30 mins. After, place it in the 65°C heating block for 10 minutes.

Take the insert out of the freezer, and add 5 μL of insert and 5 μL of vector to a new tube. Label the rest of each tube as Unligated, put the date on the tube, and stick it in the -20°C freezer incase your transformation doesn’t work. To the single tube of 10μL mix, add 10 μL of 2x Quick Ligase Buffer, and 1 μL of Quick Ligase. Let this sit at room temperature for 5 minutes.

You are now done. If you are going to transform this construction product, add all 21μL to a tube of whatever bacteria you're using.

Competent Cells

Protocol for chemically competent cells

• Grow an overnight culture
• Dilute 1/100 and grow as a 500 ml culture until OD600 reaches 0.3 – 0.5 (about 2.0 hours)
• Aliquot into ten 50 ml centrifuge tubes (ten tubes, 50 ml each).
• Chill the cells 15-20 minutes on ice (From now on always work in the cold room and have cold pipettes and tubes ready)
• Spin for 15 minutes at 4C at 3000 rpm
• Resuspend the cells in 50 ml of ice-cold 100 mM CaCl2
• Incubate for 20 minutes at room temperature
• Spin for 10 minutes at 4C at 3000 rpm
• Resuspend the cells in 1.0 ml of ice-cold 100 mM CaCl2
• Add cold sterile glycerol to a final concentration of 25% (1.0 ml of 50% glycerol and 1.0 ml of cells).
• Freeze in 100 l aliquots on dry-ice/ethanol and store at -80C

Quick Ligation

2X Quick Ligation Buffer
132 mM Tris-HCL
20 mM dithiothreitol
2 mM ATP
15% Polyethylene glycol (PEG 6000)
pH 7.6 at 25 degrees Celsius

• Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O
• Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O.
• Add 10 μl of 2X Quick Ligation Buffer and mix
• Add 1 μl of Quick T4 DNA Ligase and mix thoroughly
• Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes
• Chill on ice, then transform or store at –20°C
• Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency