Team:SDU-Denmark/protocols

= Protocols =
Any deviation from these protocols will be noted in the [Labnotes]

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:

Other programs have been used as well

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

Restriction digest

How to digest DNA using fast digest restriction enzymes.

Important remarks
Remember to load a documentation slot next to the marker and take a picture of this for later documentation.

Materials
Restriction mixture:

For 1 digest reaction.

• 24 µL H2O (or 26 µL if only one restriction enzyme is used)

• 2 µL enzyme A

• 2 µL enzyme B

• 4 µL Fast Digest green buffer

• 10 µL PCR product

Protocols
1. Prepare a purification gel

2. Mix the restriction mixture in en eppendorf tube by pipetting up and down

3. Leave for 5 min. at 37°C (no shaking!)

4. Immidiately load the restriction mixture in the gel

5. Run the gel and perform a purification step

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.

8. Store DNA at -20°C

Plasmid miniprep kit (Fermentas)

How to isolate plasmids from cultures

Important remarks
All steps should be carried out at room temperature.

Step 1 and 2 must be carried out in the micro lab.

Materials
• Resuspension solution (with RNase A)

• Lysis solution

• Neutralization solution

• Wash solution (diluted with ethanol)

• Elution solution

Protocol
1. Resuspend pelleted cells in 250 µL Resuspension solution. Resuspend completely by vortexing. Transfer the cell suspension to microcentrifuge tubes.

2. Add 250 µL Lysis solution and mix thoroughly by inverting the tube 4-6 times until the solution is viscous and slighty clear. (Do not vortex!)

3. Add 350 µL Neutralization buffer and mix immediately and thoroughly by inverting the tube 4-6 times. (It is important to mix gently to avoid localized precipitation)

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

5. Transfer supernatant to the supplied GeneJet spin column, without disturbing or transferring the white precipitate.

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

7. Add 500 µL Wash solution to the column. Centrifuge for 30-60 s. and discard the flow-through. Place column back into the same tube.

8. Repeat step 7.

9. 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)

10. Transfer the column into a fresh 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane to elute the plasmid DNA (do not touch the membrane with the pipette tip!). Incubate for 2 min. at room temperature and centrifuge for 2 min.
Optional: repeat elution step to increase the overall yield by 10-20%.

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

Preparation of Agarose for gel electrophoresis

How to prepare Agarose for gel electrophoresis.

Important remarks
Agarose concentration is dependent on the size of the DNA fragment that needs to be seperated (see the door of the incubator in the gel room)

Addition of EtBr is carried out in fume hood.

Materials
• Seachem Agarose

• TAE buffer

• EtBr

Protocol
1. For a 1% agarose gel mix 3 g agarose and 300 mL TAE buffer in a 500 mL flask.

2. The mixture is heated for 5 min. at max temperature in micro-wave. Remember to note name, date and –EtBr on the flask.

3. Place flask in the incubator for 20 min or at room temperature until cooled to 60°C.

4. Add 5 droplets of EtBr.

5. Cast gel and leave for 20 min until the gel is set. Remaining agarose solution is placed in incubater for later use.

6. Load gel and run gel. Load only 5 µL of DNA marker

Preparation of SOB and SOC media

How to prepare SOB and SOC media for transformation.

SOB medium
Used in growing bacteria for preparing chemically compotent cells.

Materials
For 1 L:
• 20 g tryptone

• 5 g yeast extract

• 0.5g NaCl

• dH2O to 1 L

• KCl (is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O)

• 2M MgCl2 (is made by dissolving 19g MgCl2 in 90 mL dH2O =>adjust to obtain a volume of 100 mL using dH2O => autoclavate)

Protocol
1. Add tryptone, yeast extract and NaCl to 950 mL of dH2O and shake until solute has dissolved.

2. Add 10 mL of 250 mM solution of KCl

3. Adjust volume to 1 L using dH2O

4. Autoclavate for 20 min.

5. Just before use add 5 mL of sterile solution of 2M MgCl2

SOC medium:
Materials
• SOB medium.

• 1M glucose (is made by dissolving 18g of glucose in 90 mL of dH2O => adjust to obtain a volume of 100 mL using dH2O)

Protocol
1. Cool SOB medium to 60°C

2. Add 20mL of 1M glucose.

Making competent cells

How to make competent cells for transformation – the iGem way

Important remarks
All of the experiment needs to be carried out in the micro lab

Materials
• SOB media (see separate protocol)

• Ice cold CCMB80 buffer

10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)

80 mM CaCl2 .2H2O (11.8g/L)

20 mM MnCl2.4H2O (4.0 g/L)

10 mM MgCl2.6H2O (2.0 g/L)

10% glycerol (100 mL/L)

Adjust pH down to 6.4 with 0.1 M HCl if nessessary.

Adjusting pH down will precipitate manganese dioxide from Mn containing solutions.

Sterile filter and store at 4°C. Slight dark precipitate appears not to affect its function.

• Top10 cells grown on SOB plate

• Glycerol

• SOC (see separate protocol)

Protocol
Preparing seed stocks.

1. Streak Top10 cells on an SOB plate and grow for single colonies at 23°C. (room temperature)

2. Pick single colonies into 2 mL of SOB medium and shake overnight at 23°C.

3. Add glycerol to 15%

4. Aliquot 1mL samples to Nunc cryotubes

5. Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 min. (this step may not be necessary)

6. Place in -80°C freezer indefinetly

Preparing competent cells
1. Inoculate 250 mL of SOB medium with 1 mL vial of seed stock and grow at 20°C to an OD600nm of 0.3. (This takes approximately 16 h.) You can adjust this temperature somewhat to fit your schedule aim for lower, not higher OD if you cannot hit this mark.

2. Cebtrifuge at 3000g at 4°C for 10 min. in a flat bottom centrifuge bottle (flat bottom centrifuge tubes make the fragile cells easier to resuspend pellets by mixing before adding large amounts of buffer).

3. Gently resuspend in 80 mL of ice cold CCMB80 buffer. (sometimes this is less than completely gentle. It still works).

4. Incubate on ice for 20 min.

5. Centrifuge again at 4°C and resuspend in 10 mL of ice cold CCMB80 buffer.

6. Test OD of a mixture of 200 µL SOC and 50 µL of the resuspended cells.

7. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.

8. Incubate on ice for 20 min.

9. Aliquot to chilled screw top 2 mL vials or 50 µL into chilled microtiter plates.

10. Store at -80°C indefinitely.

11. Optional: Test competence.

Measurement of competence

How to test transformation efficiency of competent cells – the iGEM way

Materials
• pUC19 plasmid (Invitrogen)

• SOC medium (see separate protocol)

Protocol
1. Transform 50 µL of competent cells with 1 µL of standard pUC19 plasmid. This is at 10 pg/µL or 10-5 µg/µL (This can be done by diluting 1 µL of NEB pUC19 plasmid (1 µg/µL, NEB part number NS3401S) into 100 mL of TE))

2. Keep on ice for 30 min.

3. Heat shock 60 s. at 42°C (Very important)

4. Add 250 µL SOC medium

5. Incubate at 37°C for 1 h. in 2 mL centrifuge tubes. (these tubes works well with transformation)
- Transformation with plasmids pSB1AC3 and pSB1AT3, which are chloramphenicol and tetracycline resistant, incubating for 2 h. yields many more colonies.

6. Plate 20 µL on AMP plates and spread.

7. Incabate plates at 37°C over night.

8. Count the colonies (good cells should yield around 100-400 colonies) Transformation efficiency is (dilution factor = 15) x colony x 105/µg DNA

We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

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