Difference between revisions of "Team:KU Leuven/Research/Methods"

Theory
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Protocol
Make a liquid culture of a single colony in 1-3 mL salt free LB

1. Preparation of lysate starting from stock plate of phage

Stockplate of E. coli MG1655

- O/N culture of E. coli MG1655

- Take 500 µl of O/N culture and add P1 (-80°C). Incubate O/N at 37°C

- Take single plaques of P1 stock plate and bring it in a sterile eppendorf tube together with 200 µl of MQ

- O/N extraction, shaking at 37°C

- Add 0.01, 0.1, 1, 10 and 100 µl of extraction to 500 µl of a stationary phase culture of E. coli MG1655. Vortex and bring to a sterile petri dish with LB.

- Add LB soft agar with 10 mM MgSO4 and 5 mM CaCl2 in it and incubate at 37°C.

- Take plate with best lysis.

- Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.

- Put soft agar in syringe (10 ml and G22).

- Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.

- Take 650 µl and bring in new eppendorf tube (total 1300 µl).

- Extraction with 30 µl of CHCl3

- Vortex heavy!!!

- Storage of lysate at 4°C.

2. Preparation of lysate of donor strain.

- Add to 500 µl aliquots of O/N stationary phase culture of donor strain 0.1, 1, 10 and 100 µl of lysate. First, you have to centrifugate the lysate to be sure the chloroform is at the bottom of the eppendorf tube.

- Add LB softagar with 10 mM MgSO4 and 5 mM CaCl2. Incubate at 37°C.

- Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.

- Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.

- Take 650 µl and bring in new eppendorf tube (total 1300 µl).

- Extraction with 30 µl of CHCl3

- Vortex heavy!!!

- Storage of lysate at 4°C.

3. Transduction to acceptor strain.

- Concentrate 500 µl of O/N stationary phase culture of acceptor strain 5 times in LB with 10 mM MgSO4 and 5 mM CaCl2.

- Add 0.1, 1, 10 and 100 µl of the donor strain lysate to 100 µl aliquots of the acceptor strain.

- Incubate 30 minutes at 37°C

- Plate out on selective medium en incubate overnight

-Plate also lysate out on normal LB plate to see if lysate contains no contamination.

Theory
The Gibson assembly, as described by Gibson et al., is a rapid DNA assembly method which assures directionional cloning of fragments in one single reaction. For the Gibson assembly to happen, three essential enzymes are needed : a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. For this reaction, we used NEBbuilder. In the first step of this reaction, the exonuclease rappidly cleave off the 5’ DNA ends. These enzymes are then heat inactivated at 50 degrees. In the second step, the complementary overhangs, which have to be put in there upon desiging of the fragments, start to anneal.The polymerase then fills in the gaps. In the final step, the ligases covalently joins both ends. After this, the plasmid should be ready to be transformed. This text was based on the IDT website as seen on 13/09/2015

Figure 1

Gibson assembly reaction and its essential components E.coli

Materials

Protocols

Protocol

1. Prepare selective media (LB with 0.25% agar (2,5g/l) in Petri dishes (85 mm dia.).

2. Apply 1,5 µL of the diluted cell suspensions from mid-log-phase cultures (~2×105 cells/µL (OD=0,5)) to the center of the plates, and let them dry in air for 15 min

3. Incubate at 37 °C for 10 h.

Materials

- 1L sterile LB without NaCl (10g tryptone, 5g yeast extract per 1L)

- 500 mL of 10% v/v glycerol

- cold falcon tubes of 50 mL

- cold eppies and pipette tips

Protocol

Day 1:

1. Strike your cells on a plate and grow overnight in 37°C.

Day 2

1. Pick a single colony from your plate and grow it in 1-3 mL salt free LB overnight in 37°C.

Day 3:

1. Grow 300-400 mL cells (without salt) in 37°C untill the OD reaches 0.6 (use a starting culture).

2. Cool down on ice and from now on perform all the steps in 4°C.

3. Spin the cells down in falcon tubes (3500 g, 20 min, 4°C). Using falcon tubes ensures no detergents present.

4. Resuspend the pellet in 30 mL icecold 10% glycerol (filtered to a disposable bottle to ensure no detergents). Spin down the cells (5000 g, 10 min, 4°C). Repeat this step 3 times.

5. Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not much more than 1.5 mL).

6. Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol.

7. Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80°C.

Materials:

- DNA

- electrocompetent cells

- SOC

- ice-cold cuvettes

Protocols:

1. Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)

2. Electroporate (Eppendorf, 1700 V, 4 msec)

3. Add 950 µl of SOC solution

4. Incubate for one hour at 37 °C

5. Plate this out on pre-warmed plates (37 °C)

Materials:

overnight liquid cultures

Thermo Scientific geneJET Plasmid Miniprep Kit

Protocol :

1. Transfer the liquid cultures to an eppendorf tube and spin down a 13400 rpm for 1 à 2 minutes

2. Resuspend the pelleted cels in 250 µL of the Resuspension solution. The cells should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

3. Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.

Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA

4. Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.

Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate shoud become cloudy

5. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.

6. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate. Note. Close the bag with GeneJET Spin Columns tightly after each use!

7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

8. Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

9. Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.

10. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.

11. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.

12. Discard the column and store the purified plasmid DNA at -20°C.

Materials:

PCR mixture

Thermo Scientific GeneJET PCR Purification Kit

Protocol:

1. Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

2. Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through. Notes. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flowthrough. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use!

3. Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.

4. Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.

Note. For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended. If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column. If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.

5. Discard the GeneJET purification column and store the purified DNA at -20 °C.

Protocol :

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl). For example, add 300 µl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.

4. Place a QIAquick spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load and spin again.

5. Discard flow-through and place QIAquick column back in the same collection tube. Collection tubes are re-used to reduce plastic waste.

6. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.

7. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm). IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

Protocol:

1. Blank the nanometer with 2µL the buffer you used to elute your sample

2. put 2 µL of the sample on the nanometer, and average the result.