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:

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.