Team:Cardiff Wales/protocols

Procedures

Experimental Protocols:

1) Autoclaver Protocol

  • Fill with distilled water (upstairs room) to above grate
  • Place basket in
  • Put in beakers (anything to be autoclaved – NOT NON AUTOCLAVE PLASTIC)
  • Place lid on (align arrows) and tighten
  • Press green button.


2) Preparing LB (Luria broth) 

  •  Making up Liquid LB:
  •  Zero Balance
    • Add 10g of High Salt LB broth (RedTop, WhiteTub) to 500ml container (BlueTop)
    • Add 400ml of distilled water to (BlueTop)
    • Add autoclave tape and autoclave.
  •  Making up Agar LB
    • Zero Balance
    • Add 10g of High Salt LB broth (RedTop, WhiteTub) to 500ml container (BlueTop)
    • Add 400ml of distilled water to (BlueTop)
    • Add 4g of Agar Granules
    • Add autoclave tape and autoclave.
  •  Add antiobiotic (Chloro/Carb) following cooling
    • Carb: 100mg/ml (1000x dilution)
    • Chloro: 34mg/ml (1000x dilution)
    • E.g 1L Liquid LB-Carb requires 1ml of Carb

http://www.addgene.org/plasmid-protocols/bacterial-plates/


*Bacterial transformation: Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids, even those designed for use in mammalian cells, carry both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria. *

3) Transformation protocol: 

(KEEP ALL TUBES ON ICE)

  •  Take competent cells (NEB 10-beta competent E. coli cells) from -80degrees freezer (upstairs) and thaw on ice (around 10mins)
  • Add entire 50ul tube of competent cells to 1.5ml eppendorf tube
  • Find appropriate well (e.g. Plate 1: Well 3M), using pipette (yellow tip), pierce foil lid and resuspend DNA (pipette up and down)
  • Add 1-5ul containing 1pg-100ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. DO NOT VORTEX
  • Place the mixture on ice for 30 minutes
  • Heat shock at exactly 42 degrees for exactly 30 seconds (water bath)
  • Place on ice for 5 minutes
  •  Pipette 950ul of room temperature SOC (or Liquid LB) into the mixture
  • Place at 37degrees for 60 minutes in shaking incubator
  • Warm selection plates to 37 degrees (place in incubator)
  • Spread 50-100ul of mixture onto a selection plate (using pastelle pipette, ethanol dipping and flaming) and incubate overnight at 37degrees.

Creating a control:

  • Separate cells into 2x25ul portions (in 1.5ml eppendorf tubes), in one tube do not add plasmid DNA and in the other do add.
  • Continue above steps.

http://parts.igem.org/Help:Protocols/Transformation
http://www.addgene.org/plasmid-protocols/bacterial-transformation/

 

4) Agar Plate Preparation Protocol (Chloro/Carb)

(IN BIOSAFETY CABINET)

  •  Pour agar LB solution (Carb/Chloro) into petri dish until 2/3 full (around 30ml)
  • Leave to solidify for a few minutes.


5) PCR protocol

Prep:

  • 300ul LB liquid into 1.5ml eppendorf
  •  Using pipette, take a single colony from agar plate, pipette into mixture
  • Grow for 3 hours at 37degrees in shaking incubator
  • Using 0.5ul eppendorf tubes (10ul reaction) add following amounts:
    • 5ul – 2xBuffer
    • 1ul (of each) – Primers
    • 2ul – DNA sample
    • Water to make up mixture to 10ul.
  • PCR reaction:
    • 94degrees: 5mins
    • 94degress: 20seconds (35 cycles)
    • 55degrees: 20seconds (35 cycles)
    • 72degrees: 90seconds (35 cycles)
    • 72degrees: 10mins
    • Set at 15 degrees to finish.

6) Miniprep: ALL STEPS CARRIED OUT AT ROOM TEMPERATURE

*This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. *

  • Pellet 1-5ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature *
  •  Resuspend pelleted bacterial cells in 250ul Buffer P1 and transfer to a microcentrifuge tube
  •  Add 250ul Buffer P2 and mix thoroughly by gently inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. DO NOT VORTEX
  •  Add 350ul Buffer N3 and mix immediately and thoroughly by gently inverting the tube 4-6 times
  •  Centrifuge the 10 min at 13,000rpm. A white pellet will form
  •  Apply 800ul supernatant to the QIAprep 2.0 spin column by pipetting
  •  Centrifuge for 30-60 seconds and discard flow through
  •  Wash the spin column by adding 0.75ml Buffer PE. Centrifuge for 30-60s and discard the flow through
  •  Centrifuge for 1 min to remove residual wash buffer. (Ensure completely dry, tap spin tube on paper)
  •  Place the spin column in a clean 1.5ml eppendorf. To elute DNA, add 50ul Buffer EB to the centre of spin column. Let stand for 1 min, and centrifuge for 1 min.

Notes: 

  •  Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields.
    Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields.
  • To concentrate further: fill two 1.5ml eppendorf tubes, centrifuge, remove supernatant, resuspend one pellet with P1 then take this all up and resuspend the other pellet with this mixture.


7) Gel Electrophoresis Protocol

  • Preparing Gel:
  •  1xTAE (50x TAE stock – bluetop)
  •  1% Agarose (i.e 1g in 100ml TAE)
  •  Dissolve using microwave (when bubbles stop) – around 1min30 for 150ml
  •  Add 5ul per 100ml of agarose safeview (kept in fridge)
  •  Allow to cool (use water tap if necessary)
  •  Pour into gel plate (around 75ml) with the well comb in place
  •  Allow gel to set (20-30mins)
  • unning Samples:
  •  Place agarose gel into gel box, fill gel box with 1xTAE until the gel is covered
  •  Add 10ul of DNA sample to eppendorf tube
  •  Add 3-5ul loading dye to mixture
  •  Load a well with a ladder in the first lane of gel (8ul)
  •  Load 8ul of samples into each lane of gel
  • Place lid on gel box and plug in, run at 100V (check for bubbles)
  •  Let run until dye line ¾ down the gel (approx. 1-1.5hours).

Visualising Results:

  •  Using the Geldoc2000, place gel behind door.
  •  Click ‘Live Focus’
  •  Press ‘Trans UV’
  •  Click ‘UV mode’
  •  Adjust Iris Lens control and contrast for better visualisation
  •  Click ‘Freeze’ and ‘Print’.


8) PCR purification protocol 
*This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions *

  • Add 5x volume of DNA sample of QG buffer (orange dye) in purple spin column
  •  Centrifuge for 1 min and discard flow through
  •  Wash the spin column by adding 750ul Buffer PE. Centrifuge for 30-60s and discard flow through
  •  Centrifuge for 1 min to remove residual wash buffer
  •  Place the spin column in a clean 1.5ml eppendorf. Elute DNA add 30ul of buffer EB to the centre of spin column, let stand for 1 min and centrifuge for 1 min.


9) Hi-Fi Assembly

  • See NEB for protocols.


10) Restrictions


11) Ligations


For ligations we used the NEB Cloner

  • we normally used a 5:1 ratio of insert to vector
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