Team:Goettingen/Notebook



Notebook

LB medium

-       Add the following components

NaCl

10 g

Yeast extract

5 g

Tryptone

10 g

H2O

Add to 1 L
  • To obtain solid media add 15g/L agar.

-       Autoclave at 121 oC for 20 min.

-       The preferred antibiotic is added with the proper concentration (e.g Ampicilin is added to a final concentration of 100 µg/ml)

  • To the liquid medium antibiotic is added upon usage.
  • For the preparation of agar plates antibiotic is added after autoclaving the media and cooling it to 60 oC. 

 

Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I, PEQLAB Technologies

Select few TOP10/BL21 E.coli transformed colonies from the LB Amp plates containing the propagated transformed colonies and inoculate them into liquid LB medium (5 ml) containing preferred antibiotic of desired concentration in each tube.

Incubate the LB tubes with the transformed colonies at 37ᵒC in a shaker for about 12 – 15 hours (overnight) with agitation (150 rpm).

Extract the plasmids from the incubated LB cultures using the peqGOLD Plasmid Miniprep Kit I.

Centrifuge the culture at 10000 x g for 2 min to obtain the pellet and repeat the process until the culture is completely centrifuged. Store the pellet of 1 ml of the culture at -20ᵒC for future use.

Resuspend the pellet in 250 µl of Solution I of the Kit (which is normally kept at 4ᵒC because of the RNase) and vortex until the pellet is resuspended.

Add 250 µl of Solution II to the resuspended mixture and gently mix by inverting and rotating the tubes 6 -10 times to obtain a cleared lysate. Incubate the mixture for 2 min to obtain optimum results.

Add 350 µl of Solution III to the cleared lysate and gently mix by inverting the tubes 6 -10 times until a flocculent white precipitate is formed. Centrifuge at 10000 x g for 10 min at room temperature.

Transfer the clear supernatant to a fresh PerfectBind DNA Column in a 2 ml Collection Tube. Centrifuge the Column with the Collection Tube for 1 min at 10000 x g at room temperature. Discard the flow-through liquid.

Add 500 µl of PW Plasmid buffer to the PerfectBind DNA Column in the Collection Tube and centrifuge for 1 min at 10000 x g. Discard the flow-through.

Add 750 µl of DNA Wash buffer to the PerfectBind DNA Column in the Collection tube and centrifuge for 1 min at 10000 x g. Discard the flow-through. Repeat this step to obtain optimum results.

Place the PerfectBind DNA Column in the Collection tube and centrifuge for 2 min at 10000 x g to dry the column matrix. This step is essential to remove ethanol from the column.

Place the PerfectBind DNA Column into a fresh 1.5 ml Eppendorf tube. Add 50 µl of pre-warmed sterile deionized water directly to the binding matrix in the PerfectBind DNA Column and centrifuge for 1 min at 5000 x g to elute the DNA.

Discard the PerfectBind DNA Column and store the eluted plasmid DNA at -20ᵒC.

Check the concentration of the plasmids by using a NanoDrop and note down the values for future experiments.

Document the dates you worked on your project.

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  • Chronological notes of what your team is doing.
  • Brief descriptions of daily important events.
  • Pictures of your progress.
  • Mention who participated in what task.

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