Team:SDU-Denmark/protocols

Protocols

Colony PCR

How to amplify DNA from bacteria colonies and solutions

Important remarks

All solutions should be kept at ice until run of PCR

Materials

Premix Phu-PCR

For 1 PCR reaction:

• 5 µL Phu-buffer

• 1.5 µL 10mM dNTP mix

• 1.5 µL 10pmol/µL forward primer

• 1.5 µL 10pmol/µL reverse primer

• 0.5 µL Pfu polymerase enzyme (add just before PCR run)

• 40 µL H2O

Total vol.: 5O µL

Premix TAQ-PCR (no proofreading):

For 1 PCR reaction

• 2.5 µL 10x TAQ-buffer + MgCl2

• 0.5 µL 10mM dNTP mix

• 1.25 µL 10pmol/µL forward primer

• 1.25 µL 10pmol/µL reverse primer

• 0.25 µL TAQ polymerase enzyme (add just before PCR run)

• 19.25 µL H2O

Total vol.: 25 µL

The TAQ polymerase has no proofreading, and should therefore only be used for size determination of DNA fragments. When PCR-product is to be purified and used for further experiments always use Phu polymerase!!! Make enough premix for your number of colonies +3

Protocol

Colony PCR:

1. Select and transfer a single colony to a PCR tube with a pipette tip (afterwards use the same for plating out on plates)

2. Add all of the H2O used in the premix to the PCR tubes and place them in the microwave at full power for 2 min. with an open lid.

3. Make the premix (without water). Do not add enzyme until just before premix is added to the PCR tubes. Mix the premix by pipetting up and down (do not vortex!)

4. Add premix to each PCR tube.

5. Run PCR

PCR of DNA in solutions:

1. Transfer 1-2 µL of DNA to each PCR tube (to obtain the correct total volume adjust the volume of the H2O)

2. Make the premix(do not add enzyme until just before premix is added to the PCR tubes). Mix the premix by pipetting up and down (do not vortex!)

3. Add premix to each PCR tube.

4. Run PCR

PCR program:

Making competent cells of E. coli for transformation

How to make competent cells for transformation

Compotent cells enough for 12 transformations

Important remarks

Use 2 ml eppendorf tubes

Cool eppendorf tubes at 4°C prior to use

Cool 50 ml 50 mM CaCl2 at 4°C prior to use

Materials

• ON culture of TOP10 cells in LB media

• Ice cold 50mM CaCl2

• LB media (pre-heated to 37°C)

Protocol

1. Dilute the culture to OD550 = 0,02 in 110 ml of LB. Incubate at 37°C with shaking until OD550 reaches 0.5

2. Divide the cells in 2x55 ml and transfer to Falcon tubes (the can hold only 55 ml). From now on proceed with the 2 tubes in parallel

3. move the CaCl2 to -20°C

4. Harvest cells by centrifugation at 4100 rpm (2160 G) at 4°C for 10 min.

5. Discard the supernatant (keep the cells on ice!)

6. Resuspend cells gently in 5 ml ice cold CaCl2 (50 mM) taken from -20°C and kept on ice.

7. Repeat the centrifugation step.

8. Discard the supernatant and resuspend cells in 1.2 ml of icecold CaCl2 (keep the cells on ice!)

9. Leave the cells on ice for 30 min => now the cells are ready for transformation.

Transformation

How to transform compotent cells

Important remarks

Pre-heat LB media to 37°C

Pre-dry LA plates with the appropriate antibiotics.

Pre-cool 2 mL eppendorf tubes.

Keep cells on ice at all times!!

Remember controls:

Positive control with your uncut vector

Negative control with no DNA

Materials

• Freshly made compotent E. coli cells

• LA plates with appropriate antibiotics

• LB media

Protocol

1. Transfer 5 µl DNA (plasmid or ligation mix) to precooled eppendorf tubes.

2. Transfer 200 µl of cells with to the tube and mix by pipetting up and down (keep the cells on ice at all times)

3. Leave on ice for 30 min.

4. Heat shock for 90 sec. at 42°C in a water bath, do not shake tubes.

5. Place on ice for 2 min.

6. Add 1.5 mL of preheated LB media (37°C)

7. Incubate at 37°C for 1 hour with gentle shaking.

8. Plate 2 plates with 150 µl mixture on LA plates with the appropriate antibiotics.

9. Pellet the remaining cells 5 min at 3500 rpm and discard approximately 900 µl of the supernatant.

10. Resuspend cells and plate out on LA plates with appropriate antibiotics.

11. Incubate all plates ON at 37°C

Ligation

How to assemble DNA biobricks

Materials

Ligation mixture:

For 1 ligation reaction

• 2 µL 10x T4 ligase buffer

• 1 µL T4 ligase (add last!)

• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks

Protocol

1. Prepare the ligation mixture and mix by pipetting up and down

2. Leave the mixture over-night at 17°C

3. Test ligation using TAQ-PCR and run test gel afterwards in order to check that the PCR product has the right size

DNA extraction from gel (fermentas)

How to extract and purify DNA from gel

Important remarks

All steps should be carried out at room temperature. All centrifugations should be carried out in a table-top microcentrifuge at >12000x g

Materials

• Binding buffer

• Wash buffer (diluted with ethanol)

• Elution buffer

Protocol

1. Weigh a 1.5 µL tube

2. Excise gel slice containing the DNA fragment using a clean scalpel (cut as close to the DNA as possible)

3. Place the gel slice into the pre-weighed tube and record the weight of the gel slice

4. Add 1:1 volume of binding buffer to the gel slice (e.g. add 100 µL of binding buffer for every 100 mg of agarose gel)

5. Incubate the gel mixture at 50-60°C for 10 min. or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes. The color of the solution should be yellow. If the color of the solution is orange or violet add 10 µL 3M sodium acetate , pH 5.2 and mix. The color will then turn yellow.

6. Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube

7. Add 700 µL of Wash buffer to the column. Centrifuge for 1 min. Discard flow-through and place the column back into the collection tube

8. Centrifuge the empty column for an additional 1 min. to completely remove residual Wash buffer This step is essential to avoid residual ethanol in the purified DNA solution

9. Transfer the column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane. Centrifuge for 1 min.

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

Genomic DNA purification

How to extract and purify genomic DNA

Important remarks

All steps should be carried out at room temperature

Be sure to mix thoroughly when adding the solutions

Addition and removal of chloroform should be carried out in fume hood

Materials

• Lysis solution

• Chloroform

• Precipitation solution (80 µL is diluted in 720 µL of H2O just prior to use)

• 1.2M NaCl solution

• Ice cold ethanol (70%)

• H2O

Protocol

1. Mix 200 µL of sample (ON culture) with 400 µL of Lysis solution and incubate at 65°C for 5 min.If a frozen sample is used lysis solution should be added before thawing and incubated at 65°Cfor 10 min. with occasional inverting the tube.

2. Immediately add 600 µL of chloroform, gently emulsify by inversion (3-5 times) and centrifuge the sample at 10.000 rpm for 2 min.

3. Prepare precipitation solution

4. Transfer the upper aqueous phase containing DNA to a new tube and add 800 µL of freshly prepared precipitation solution, mix gently by several inversions at room temperature for 1-2 min. and centrifuge at 10.000 rpm for 2 min.

5. Remove supernatant completely (do not dry) and dissolve DNA pellet in 100 µL of 1.2M NaCl solution by gentle vortexing (make sure that the pellet is completely dissolved) To avoid loosening the pellet, keep the tube in the same angle as when placed in the centrifuge!

6. Add 300 µL of cold ethanol, let the DNA precipitate (10 min. at -20°C) and spin down (10.00 rpm, 3-4 min.).Pour off the ethanol and dissolve DNA in 15 µL of sterile dH2O by gentle vortexing.

7. Measure DNA concentration on nanodrop.