Difference between revisions of "Team:Cardiff Wales/protocols"

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<h3 align="left"> PCR </h3>
 
<ul>
 
<ul>
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<li></li>
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<li>For a 20 µl PCR reaction, add 10 µl PCR Super Master Mix/Taq Buffer, 1 µl forward primer, 1 µl of backward primer, 6 µl autoclaved water and 2 µl Mini-prepped DNA, ensuring PCR products in are in suitable PCR tubes </li>
 +
<li>Place the PCR tubes into the PCR machine and run PCR at a temperature and duration appropriate for the primers to work effectively</li>
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<li> Once finished, add 6 µl of PCR product to individual wells on a gel (See Agarose Gel Electrophoresis) </li>
 
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</ul>
 
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<h3 align="left"> Transformation of <i> E. coli </i> cells </h3>
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<h3 align="left"> Agarose Gel Electrophoresis </h3>
 
<ul>
 
<ul>
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<li></li>
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<li>Add 5 ml of 50x TAE (Tris-acetate-EDTA) buffer to 245 ml of distilled water</li>
 +
<li>Take 100 ml of 2% TAE Stock from step 1 and add to a 250 ml conical flask</li>
 +
<li>Add 1 g Agarose to 100 ml TAE buffer and microwave for 1 min 50 sec, making sure all agarose has dissolved</li>
 +
<li>Cool solution before adding 5 µl Safe View and mix before pouring into gel cassette. Add appropriate number of combs to the cassette, depending on the number of PCR reactions</li>
 +
<li>Leave 1% agarose gel to set for 30 min before removing the comb(s). Place set gel into the gel electrophoresis tank and add remaining 2% TAE stock to the tank, making sure the gel is covered by 2% TAE buffer</li>
 +
<li>Add 6 µl hyperladder suitable for the DNA length to the first well and add the remaining PCR reaction mixtures to the wells</li>
 +
<li>Set the voltage to 120V and run the gel for 30 minutes</li>
 +
<li>Once fully run, turn off the voltage and view the DNA bands using a UV illuminator</li>
 +
 
 
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<h3 align="left"> Transformation of <i> E. coli </i> cells </h3>
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<h3 align="left"> Transformation of Agrobacterium </h3>
 
<ul>
 
<ul>
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<li></li>
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<li>Add 100 µl Agrobacterium to 5 µl DNA to a 1.5 ml Eppendorf tube and leave on ice for 10 min</li>
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<li>Freeze in liquid nitrogen for 5 min before incubating at 37 °C for 5 min</li>
 +
<li>Place the mixture back in ice for another 5 min before adding 1 ml LB broth with no antibiotic</li>
 +
<li>Shake mixture at 28 °C for 2 hr before plating onto LK-Rif plates. Incubate these plates at 28 °C and leave for 2 days</li>
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Revision as of 14:59, 8 September 2017




Our Protocols


Liquid Broths

  • Fill a container with 400ml distilled water
  • Add 25 g/L of LB broth to the distilled water
  • Swirl the container with the water and LB broth together so that the powder has nearly dissolved
  • Autoclave the container and store in fridge at 4 °C until LB broth is required

Agar Plates (8)

  • Fill a container with 200 ml distilled water
  • Add 25 g/L of LB broth and 10 g/L Agar to the container
  • Swirl the container so that both the LB broth and Agar are nearly dissolved and autoclave
  • Add 200 µl of antibiotic (Amp, Chl, Kan etc.) to the autoclaved container along with 100 µl X-gal and 100 µl IPTG
  • Distributed finalised mixture equally between 8 petri dishes, making sure all the plates are completely covered and leave to set
  • Once set, store the plates at 4 °C until required.

Transformation of E. coli cells

  • To a 1.5 ml Eppendorf tube, add 50 µl of competent cells to 10ul of DNA and leave on ice for 10 minutes. Heat shock at 42 °C for 1 min before putting the cells back on ice for 5 min. Following this, add 1 ml LB broth with no antibiotic before shaking in the incubator 1 hour at 37 °C. On a chloramphenicol, X-gal and IPTG plate, add 5-10 glass beads with 200 ul of cells and DNA from incubation. Shake the plates until the cells and DNA are equally distributed across the plate. Remove the beads and incubate the plate at 37°C overnight.
  • If the colour of the colonies is indecipherable after incubation, place in 4 °C fridge until colour develops.

Growing Colonies

  • From plate showing successfully transformed white colonies, pick the appropriate colonies using a pipette tip and place into 5ml LB broth with suitable antibiotic (Amp, Chl, Kan etc.) Resuspend the colony in the LB broth and antibiotic before vortexing for 10 seconds. Incubate tubes at 37°C in the shaking machine overnight.

QIAprep® Spin Miniprep Kit

  • Place Buffer EB (for step 10) into 55 °C water bath. Pellet 1-5ml bacterial overnight culture by centrifugation at 13,000 rpm for 3 min at room temperature (15-25 °C)
  • Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
  • Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min
  • Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  • Centrifuge for 10 min at 13000 rpm in a table-top microcentrifuge
  • Apply 800 µl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through
  • Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through
  • Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through
  • Centrifuge for 1 min to remove residual wash buffer. After this, tap the QIAprep 2.0 spin column on the bench before centrifuging again for 1 min
  • 10) Place the QIAprep 2.0 spin column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30-50 µl Buffer EB to the centre of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min

Jena Bioscience: Fast-n-Easy Plasmid Mini-prep Kit

  • Harvest the bacterial cell culture (1-3 ml) by centrifugation
  • Resuspend pelleted bacterial cells in 300 µl lysis buffer by pipetting
  • Add 300 µl of Neutralization buffer (containing RNase A) to sample and mix gently by inverting the tube 4-6 times (do not vortex!)
  • Centrifuge at 10,000 g for 5 min at room temperature in a microcentrifuge
  • The colour of the binding mixture should change to bright yellow indicating a pH of 7.5 required for optimal DNA binding. An orange or violet colour shows a pH >7.5 and indicates and inefficient DNA absorption. In this case, it is recommended to adjust the pH of the mixture by addition of a small volume of 3 M sodium acetate, pH 5.0 before proceeding
  • Place a binding column into a 2 ml collection tube
  • Add 100 µl of Activation buffer into a Binding Column
  • Centrifuge at 10,000 g for 30 sec in a microcentrifuge
  • Apply the supernatant from step 2 into the activated Binding Column by decanting or pipetting
  • Centrifuge at 10,000 g for 30 sec
  • Discard the flow-through
  • Place the DNA loaded Binding Column into the used 2 ml tube
  • Apply 500 µl of Washing Buffer (containing Ethanol) to the Binding Column
  • Centrifuge at 10,000 g for 30 sec and discard the flow-through
  • Add 700 µl of Washing Buffer to the Binding Column
  • Centrifuge at 10,000 g for 30 sec and discard the flow-through
  • Centrifuge again for 2 min to remove residual Washing Buffer
  • Place the Binding Column into a clean 1.5 ml microtube
  • Add 30-50 µl Elution Buffer or dd-water to the centre of the column membrane
  • Incubate for 1 min at room temperature
  • Centrifuge at 10,000 g for 1 min to elute DNA

PCR

  • For a 20 µl PCR reaction, add 10 µl PCR Super Master Mix/Taq Buffer, 1 µl forward primer, 1 µl of backward primer, 6 µl autoclaved water and 2 µl Mini-prepped DNA, ensuring PCR products in are in suitable PCR tubes
  • Place the PCR tubes into the PCR machine and run PCR at a temperature and duration appropriate for the primers to work effectively
  • Once finished, add 6 µl of PCR product to individual wells on a gel (See Agarose Gel Electrophoresis)

Agarose Gel Electrophoresis

  • Add 5 ml of 50x TAE (Tris-acetate-EDTA) buffer to 245 ml of distilled water
  • Take 100 ml of 2% TAE Stock from step 1 and add to a 250 ml conical flask
  • Add 1 g Agarose to 100 ml TAE buffer and microwave for 1 min 50 sec, making sure all agarose has dissolved
  • Cool solution before adding 5 µl Safe View and mix before pouring into gel cassette. Add appropriate number of combs to the cassette, depending on the number of PCR reactions
  • Leave 1% agarose gel to set for 30 min before removing the comb(s). Place set gel into the gel electrophoresis tank and add remaining 2% TAE stock to the tank, making sure the gel is covered by 2% TAE buffer
  • Add 6 µl hyperladder suitable for the DNA length to the first well and add the remaining PCR reaction mixtures to the wells
  • Set the voltage to 120V and run the gel for 30 minutes
  • Once fully run, turn off the voltage and view the DNA bands using a UV illuminator

Transformation of Agrobacterium

  • Add 100 µl Agrobacterium to 5 µl DNA to a 1.5 ml Eppendorf tube and leave on ice for 10 min
  • Freeze in liquid nitrogen for 5 min before incubating at 37 °C for 5 min
  • Place the mixture back in ice for another 5 min before adding 1 ml LB broth with no antibiotic
  • Shake mixture at 28 °C for 2 hr before plating onto LK-Rif plates. Incubate these plates at 28 °C and leave for 2 days