Duedu:Protocols

From OpenWetWare

Jump to: navigation, search

Home        Research        Teaching        People        Publications        Vacancies        Contact        Photos        Protocols       



Contents

DNA extraction from whole blood using Chelex 100 resin

Materials

Chelex 100 Resin (Molecular Biology Grade) Microcentrifuge tubes

Prepare 10% of Chelex 100 suspension. This can be done by dissolving 2g of Chelex 100 in 20 ml of distilled autoclaved water.

Procedure

Label all tubes before proceeding.

  • 0. Set the waterbath to 99 degrees (step 4) for the incubation step
  • 1. Mix the blood well. Use the blood rotor.
  • 2. Pipette 100 ul of whole blood into a microcentrifuge tube.
  • 3. Add 200 ul of 10% Chelex 100 suspension
  • 4. Vortex the mixture and incubate at 99 degrees for 10 minutes.
  • 5. Centrifuge the mixture at high speed (13,000 xg) for 10 minutes
  • 6. Transfer the supernatant into a clean sterile 1.5 ml microcentrifuge tube and store at -20 degrees. Alternatively, You can concentrate or further clean the extracted DNA by following the procedure for purifying DNA from solution.


DNA extraction from stool using Chelex 100 resin

The first step of this protocol involves simultaneous lysis of stool with lysis buffer TEN-9 described by Abbaszadegan et al 2007 (Iranian Biomedical Journal 11 (3): 203-208) and separation of unwanted and potential inhibitors using Chelex 100, an ion chelator which limits destruction of the DNA by inactivating nucleases and chelating heavy metals that may damage DNA. The mixture is centrifuged and the supernatant containing the DNA is taken to the next stage where it is purified and concentrated from solution using silica beads. The combination of chelex extraction and purification by silica beads ensures the purest form of DNA. Incubation at 99 degrees ensures microbial DNA difficult to lyse like gram positives are efficiently lysed.


Materials

Chelex 100 Resin (Molecular Biology Grade) Microcentrifuge tubes Tris-HCL (Trizma base and HCL or Tris-HCL) EDTA NaCl SDS

Prepare 40% of Chelex 100 suspension. This can be done by dissolving 4g of Chelex 100 in 10 ml of distilled autoclaved (or nuclease free) water.

Prepare stool lysis buffer (TEN-9) containing Tris-HCL, 0.5 M; EDTA, 20 mM; NaCl, 10 mM; SDS, %0.1; pH 9.0

Procedure

Label all tubes before proceeding.

  • 1. Weigh 150-250 mg of stool into a 2 mL microcentrifuge tube and place the tube on ice. If the sample is liquid, pipette 200 uL in to the microcentrifuge tube. You may have to cut the end of the pipette tip to make it easier to get the sample. Keep the sample on ice. (check ?degradation of DNA when frozen samples are thawed - ref Qiagen stool kit handbook)
  • 2. Add 1.4 mL of TEN-9 and vortex for one minute or until the stool sample is thoroughly homogenized.
  • 3. Incubate the mixture at 95 degrees for 10 minutes (lysis of bacteria and other difficult to lyse cells)
  • 4. Vortex for 15 s
  • 5. Centrifuge at max speed (or 13,000 xg) for 15 minutes to remove particulate substances
  • 6. Transfer supernatant (about 12 mL) to a fresh 2 mL microcentrifuge tube and discard the pellet. (Place the tube with pellet into the tube recycle bottle with bleach).
  • 7. Add 200 uL of 40% Chelex 100 and incubate at 95 degrees for 5 minutes (NB. heating at this stage may not be necessarily. RT incubation may be all that is needed for chelex to inactivate nucleases, etc.)
  • 8. Centrifuge at max speed (or 13,000 xg) for 3 minutes to pellet inhibitors and chelex.
  • 9. Pipette the supernatant into a fresh 1.5 mL microcentrifuge tube. This supernatant may be used directly in PCR. Ensure to optimize the concentration of DNA and quantity of supernatant needed for the PCR.



Purifying a PCR product or Concentrating DNA from Solution

 See the original version of this protocol by Prof. Chris E. French, University of Edinburgh

If your PCR product looks pure enough to continue with (ie a single band of the expected size, with no extra unwanted DNA bands), you can purify it directly from the remaining 45 microlitres of the reaction mixture as follows.


Materials required

6 M sodium iodide. Weigh out 18 g of sodium iodide and add water to give a volume of 20 ml. Not very much water is required (about 15 ml). The sodium iodide may take some time to dissolve. Safety note: sodium iodide was formerly listed as ‘highly toxic’. It has now been reclassified as ‘harmful’, but treat it with caution. Wear gloves. The solution should be stored at 4˚C. It will gradually turn yellow over time, but this does not seem to have any adverse effect on the procedure.

Glass bead suspension. Weight out 1 g of silica beads (eg Sigma S-5631, mixture of particle sizes from 0.5 to 10 micrometres) into a universal tube. Safety note: silica powder is irritating to the eyes and lungs. Wear eye protection and a dust mask when working with dry silica powder. To remove the finest particles, suspend the beads in 10 ml PBS (phosphate buffered saline; I use 0.21g KH2PO4; 0.726g Na2HPO4; 9g NaCl; Water 1L). In order to remove the finest particles, spin down the mixture at 500 xg for 1 minute and remove the supernatant. Repeat two more times. Spin the pellet at 1000 xg for 1 minute to remove excess liquid and resuspend the beads in 5 ml of 3 M sodium iodide: See safety note above for sodium iodide. The suspension should be stored at 4˚C. The glass beads must be vigorously resuspended before use.

Wash Buffer: 10 mM Tris/HCl, pH 7.5, 2.5 mM EDTA, 50 mM NaCl, 50% v/v ethanol. To make 100 ml, weight out:

  • Tris base: 24 mg
  • Tris.HCl: 127 mg
  • NaCl: 293 mg
  • Na2EDTA: 93 mg

Dissolve in 50 ml water. Add 50 ml absolute ethanol. Store at -20˚C (it will not freeze due to the high alcohol content). This solution is stable at room temperature but must be ice-cold when used.

EB (Elution Buffer): This is 10 mM Tris, pH 8. To make 100 ml of EB, dissolve 53 mg Tris base and 88 mg Tris hydrochloride in 100 ml water. Sterilise by autoclaving.



Procedure

Purifying PCR products from solution

  • 0. Turn on a waterbath to 55˚C so that it has time to warm up before you need it in step 11.
  • 1. Transfer 40 to 45 microlitres of PCR reaction mixture from the 0.5 ml PCR tube to a 1.5 ml microcentrifuge tube.
  • 2. Add 150 microlitres of 6 M sodium iodide.
  • 3. Add 20 microlitres of glass bead suspension. Note: mix the suspension well before removing the glass beads.
  • 4. Mix and incubate on ice for 15 minutes (or longer; the exact time is not important). If all goes well, the DNA will stick to the glass beads.
  • 5. Spin the tube briefly in a microcentrifuge to get the beads to the bottom of the tube. The speed is not critical, and 30 seonds or so should be plenty, since glass beads settle very well.
  • 6. Remove the supernatant to a waste beaker. Ultimately this can be discarded to the drains.
  • 7. Add 250 microlitres of ice-cold wash buffer. (This is stored in the freezer and should be kept on ice when out of the freezer). Mix by inversion. Do not attempt to resuspend the glass beads. Spin the tube briefly and remove the supernatant to your waste beaker. Remove as much of the supernatant as possible. The purpose of this step is to wash away sodium iodide.
  • 8. Repeat step seven twice more.
  • 9. If there is still some liquid stuck to the sides of the tube, spin again briefly and remove it. Make sure that you have removed as much of the liquid as you possibly can.
  • 10. Add 40 microlitres of EB (elution buffer). Resuspend the glass beads by pipetting up and down. Make sure that they are well resuspended.
  • 11. Incubate in a 55˚C waterbath for 10 minutes or so, mixing again by flicking the tube at the 5 minute mark (this is probably not essential, but the beads do tend to settle very quickly).
  • 12. Spin at high speed for 1 minute.
  • 13. Transfer the supernatant to a clean labelled tube. Try to avoid getting any glass beads in it. Discard the tube with the glass beads.
  • 14. The DNA should be stored at -20˚C. You can check that the purification procedure has worked properly by running another 5 microlitres of the DNA on a gel, as before.


Purifying (and concentrating) DNA from Solution

After using the Chelex 100 method of extracting DNA, you will usually have a large volume of extracted DNA with concentration not so high. In order to concentrate, you will need to bind the extracted DNA to silica, wash and then elude in a smaller volume. Alternatively, you may just used any PCR purification kit (expensive) following the manufacturer's protocol.

The process here is an adaptation of the method for purifying PCR products from solution.

  • 0. Turn on a waterbath to 55˚C so that it has time to warm up before you need it in step 11.
  • 1. Transfer 200 to 250 microlitres of extracted DNA from the supernatant of Chelex 100 extraction reaction to a 1.5 ml microcentrifuge tube.
  • 2. Add 3 times volume from step 1 above of 6 M sodium iodide.
  • 3. Add 50 microlitres of glass bead suspension. Note: mix the suspension well before removing the glass beads.
  • 4. Mix and incubate on ice for 15 minutes (or longer; the exact time is not important). If all goes well, the DNA will stick to the glass beads.
  • 5. Spin the tube briefly in a microcentrifuge to get the beads to the bottom of the tube. The speed is not critical, and 30 secs or so should be plenty, since glass beads settle very well. You may spin for 10 t0 15 secs using the quick spin microfuge.
  • 6. Remove the supernatant to a waste beaker. Ultimately this can be discarded to the drains.
  • 7. Add 400 microlitres of ice-cold wash buffer. (This is stored in the freezer and should be kept on ice when out of the freezer). Mix by inversion. Do not attempt to resuspend the glass beads. Spin the tube briefly and remove the supernatant to your waste beaker. Remove as much of the supernatant as possible. The purpose of this step is to wash away sodium iodide.
  • 8. Repeat step 7 twice more.
  • 9. If there is still some liquid stuck to the sides of the tube, spin again briefly and remove it. Make sure that you have removed as much of the liquid as you possibly can.
  • 10. Add 50 microlitres of EB (elution buffer). Resuspend the glass beads by pipetting up and down. Make sure that they are well resuspended.
  • 11. Incubate in a 55˚C waterbath for 10 minutes or so, mixing again by flicking the tube at the 5 minute mark (this is probably not essential, but the beads do tend to settle very quickly).
  • 12. Spin at high speed for 1 minute.
  • 13. Transfer the supernatant (containing your DNA) to a clean labelled tube. Try to avoid getting any glass beads in it. Place the tube with the glass beads into the microtube recycle container.
  • 14. The DNA should be stored at -20˚C. You can check that the purification procedure has worked properly by running 5 microlitres of the DNA on a gel.




Purifying DNA from a Gel

If your PCR has multiple products, you may need to purify it from a gel.

  • 1. Run the gel, stain with Gel Green and visualize it on the blue light transilluminator (see the page on Gel Electrophoresis).
  • 2. Cut out the band, with as little total gel as possible, and put it in a preweighed microfuge tube, then reweigh it to determine the weight of the gel in mg.
  • 3. Add three times that amount of 6 M NaI (eg, if 100 mg, add 300 microlitres of NaI).
  • 4. Place the tube in the 55 C waterbath for 5 minutes, mixing about half way through. Check that the gel has fully melted. If not, give it another minute or so. (Note - this works fine with ordinary agarose, you don't need special low-melting agarose).
  • 5. Add 5 microlitres of glass bead suspension and incubate on ice (step 4 above), then continue with the protocol as described above.


Making your own electrocompetent cells

This protocol has been copied directly from the New England Biolabs website (Link will take you to an external site. If you want to stay here, right click and open in a new tab or page).

It is important that you optimize it for your own application and strains.

There is an interactive version of the protocol as well.

Media

SOB 2% tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4

SOC SOB + 20 mM glucose



Antibiotics for Plasmid selection

Antibiotic Working Concentration Ampicillin 100 µg/ml Carbenicillin 100 µg/ml Chloramphenicol 33 µg/ml Kanamycin 30 µg/ml Streptomycin 25 µg/ml Tetracycline 15 µg/ml Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).


Procedure (for 2, 250 ml cultures)

  1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm.
  2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks.
  3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
  4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.

Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.

  1. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
  2. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
  3. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
  4. At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months.


Electroporation Protocol

The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.

  1. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
  2. Place recovery SOC in 37°C water bath.
  3. Pre-warm LB-antibiotic plates at 37°C.
  4. Thaw cells on ice for 10 min or use freshly made cells.
  5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
  6. Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
  7. Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.

Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).

  1. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
  2. Rotate in the 37°C incubator for 1 h.
  3. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
  4. Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
  5. Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
  6. Incubate overnight at 37°C.

Calculation

If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)

cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)
Personal tools