Team:Goettingen/Experiments



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.

 

Plasmid Extraction - using QIAprep Spin Miniprep Kit (QIAGEN)

This protocol describes the purification of plasmid DNA from 5 ml overnight cultures of E. coli grown in LB medium using the QIAprep Spin Miniprep Kit. (QIAGEN).

  • Add the provided RNase A solution to buffer P1, mix, and store at 4 oC.
  • Add ethanol (96–100%) to Buffer PE before use.
  • All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.

 

- Add the proper antibiotic with the proper concentration to 5 ml LB medium (e.g Ampicilin is added to a final concentration of 100 µg/ml).

- Inoculate the medium with the desired E.coli strain and incubate overnight at 37 oC with agitation (150 rpm).

- Pellet the overnight culture by centrifugation at 8000 rpm (6800xg) for 3 min at room temperature.

- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer it to a microcentrifuge tube.

- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. 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 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

- Apply the supernatant to the QIAprep spin column by decanting. Centrifuge 60 s. Discard the flow-through.

- Wash the QIAprep spin column by adding 500 μl Buffer PB and centrifuging for 60 s. Discard the flow-through.

- Wash QIAprep spin column by adding 750 μl Buffer PE and centrifuging for 30–60 s.

- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

- Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.

- To elute DNA, add 50 μl water (40 – 60 oC) to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

 

Blunt End Ligation in pJET1.2 vector –Clone JET PCR Cloning Kit–Thermo Scientific

 

-       Set up the following componentson ice for the ligation reaction.

Components

Volume

2x Reaction buffer

10µl

Blunt end PCR amplified cellulase gene

125ng

pJET 1.2 Plasmids

50ng (1µl)

T4 DNA Ligase

1µl

Water(RNase free)

xµl

Total Volume

20µl

-       Vortex briefly and centrifuge for 3-5 sec at 5000 rpm.

-       Carry out the ligation at 20°C for 20 min at room temperature (22-25°C) and then place in ice for 5 min and use for transformation immediately or store at -20°C.

Cloning principle:

  • pJET1.2 is a linearized cloning vector which accepts inserts from 6 bp to 10 kb. Since the 5’-ends of the vector contain phosporyl groups, therefore phosphorylation of the PCR products is not required.
  • Optimal insert/vector ratio is 3:1. (0.15 pmol ends of insert and 0.05 pmol ends of vector)
  • Optimal PCR product quantity for ligation reaction is to be calculated from the Kit protocol, For the length of 2500 bp of PCR product, to have 0.15 pmol ends of the PCR product in the ligation reaction 125 ng of the PCR product should be used.
  • For PCR products more than 3 kb, ligation can be prolonged to 30 min. The cellulose gene is approximately 2.5 kb so, ligation can be done for about 20 min.

 

Transformation of pJET1.2 vector with cellulase gene into TOP10 E.coli cells:

-       Thaw the chemically competent TOP10 E.coli cellsand then add to the prepared ligation mixture. (1 aliquot of E.coli cells/ ligation mixture)

-       Incubate the mixture on ice for 15 min and then heat shock the cells at 42°C for 45-50 sec and then place on ice for 5 min.

-       Add 500µl of LB medium to the transformation mixtureand incubate at 37°C for 1 hour in shaker.

-       Propagate 50µl of the transformation mixture on a LB Amp plate and centrifuge the remaining cells at 13000rpm for 1 min and remove the supernatant and then re-suspendthe pellet in the remaining supernatant and then propagate in a new LB Amp plate.

-       Incubate the propagated plates at 37°C for about 12 – 15 hours (overnight).

 

 

Experiments & Protocols

Describe the experiments, research and protocols you used in your iGEM project.

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