First step is centrifugation to collect the cells. Having this pellet, you can use any standard protocol that works well in your hands for example for saliva samples.

Many of the commercial kits are based on guanidinium thyocyanate / silica extraction methods for which noncommercial protocols are available. Once you pellet the cells you might be able to lyse them in water and use an aliquot directly in PCR without going through a full blown extraction protocol.

You can do it straight from urine as as either from small urine volume using Norgen Biotek's Urine DNA Isolation Micro Kit (Cat# 18100) or from large urine volumes (up to 25mL urine) using Norgen Biotek's Urine DNA Isolation Kit (Slurry Format) Cat# 48800

With standard extraction kits its easy to extract, what I need is the manual protocol which is not costly and one can extract a lot of samples. Like I had extracted DNA from Saliva the other day without using a standard protocol. There will be changes in few steps of extraction and some chemicals will be used extra like DTT and the ingredients of lysis buffer, do you guys have experience with these DTT use and at point we should use it? Do we have to use it along with Proteinase K?

Buffers and Solutions

Please see Appendix 1 for components of stock solutions, buffers, and reagents.

Dilute stock solutions to the appropriate concentrations.

Ammonium acetate (10 M) (used as an alternative to dialysis, Step 9)

Dialysis buffer (used as an alternative to ethanol precipitation, Step 9)

50 mM Tris-C1 (pH 8.0)

10 mM EDTA (pH 8.0)

Prepare four lots of 4 liters of dialysis solution and store at 4°C.

Ethanol (used as an alternative to dialysis, Step 9)

Lysis buffer

10 mM Tris-CI (pH 8.0)

O.I M EDTA (pH 8.0)

0.5% (w/v) SDS

20 pglml DNase-free pancreatic RNase

The first three ingredients of the lysis buffer may be mixed in advance and slored at room temperature.

Rase is added to an appropriate amount of the mixture just before use. Adding Rase to the lysis buffer eliminates the need to remove RNA from semipurified DNA at a later stage in the preparation.

Pancreatic RNase is not highly active in the presence of 0.5% SDS, but when added at high concentrations,

it works weB enough to degrade most of the ceBular RNA.

Phenol, equilibrated with 0.5 M Tris-C/ (pH 8.0)

.4 IMPORTANT The pH of the phenol must be -8.0 to prevent DNA from becoming trapped at the

interface between the organic and aqueous phases (please see Appendix 8).

TE (pH 8.0)

Tris-buffered saline rrBS)

Enzymes and Buffers

Proteinase K (20 mg/m/)

For this protocol, we recommend the use of a genomic grade proteinase K that has been shown to be free

of DNase and RNase activity. Please see Appendix 4.

Gels

Pulsed-field gel (please see Chapter 5, Protocols 17 and 18) or Conventional horizontal 0.6% agarose gel

After lysis of urine

Treatment of Lysate with Proteinase K and Phenol

1. Transfer the lysate to one or more centrifuge tubes that fit into a Sorvall SS-34 rotor, or equivalent.

The tubes should not be more than one-third full.

2. Add proteinase K (20 mg/ml) to a final concentration of 100 ~g/ml. Use a glass rod to mix the enzyme solution gently into the viscous lysate of cells.

3. Incubate the lysate in a water bath for 3 hours at 50°C. Swirl the viscous solution from time to time.

4. Cool the solution to room temperature and add an equal volume of phenol equilibrated with 0.1 M Tris-Cl (pH 8.0). Gently mix the two phases by slowly turning the tube end-over-end for 10 minutes on a tube mixer or roller apparatus. If the two phases have not formed an emulsion at this stage, place the tube on a roller apparatus for I hour.

Blin and Stafford (1976) recommend the use of 0.5 M EDTA (pH 8.0) in the lysis buffer. However, the density of the buffer almost equals that of phenol, which makes separation of the phases difficult.

The lysis buffer used here contains EDTA at a concentration of 0.1 M, which permits easier separation of the phenolic and aqueous phases while maintaining a high degree of protection against degradation of the DNA by nucleases and heavy metals.

5. Separate the two phases by centrifugation at 5000g (6500 rpm in a Sorvall SS-34 rotor) for 15 minutes at room temperature.

6. Use a wide-bore pipette (O.3-cm diameter orifice) to transfer the viscous aqueous phase to a fresh centrifuge tube.

When transferring the aqueous (upper) phase, it is essential to draw the DNAinto the pipette very slowly to avoid disturbing the material at the interface and to minimize hydrodynamic shearing forces. If the DNA solution is so viscous that it cannot easily be drawn into a wide-bore pipette, use a long pipette attached to an aspirator to remove the organic (lower) phase as follows:

i. Beforestarting, make sure that the vacuum traps are empty and secure, so that phenol cannot enter the vacuum system.

II. With the vacuum line closed, slowly lower the pipette to the bottom of the organic phase.

Wait until the viscous thread of aqueous material detaches from the pipette, and then carefully open the vacuum line and gently withdraw all of the organic phase. Close the vacuum line and quickly withdraw the pipette through the aqueous phase. Immediately open the vacuum line to transfer the residual phenol into the trap.

iii. Centrifuge the DNAsolution at 5000g (6500 rpm in a Sorvall SS-34rotor) for 20 minutes at room temperature. Protein and clots of DNA will sediment to the bottom of the tube.

Transfer the DNAsolution (the supernatant) into a 50-ml centrifuge tube, leaving behind

the protein and clots of D A.

7. Repeat the extraction with phenol twice more and pool the aqueous phases.

8. Isolate DNA by one of the following two methods.

To ISOlATE DNA IN THE SIZE RANGE OF 150-200 KB

a. Transfer the pooled aqueous phases to a dialysis bag. Close the top of the bag with a dialysis tubing clip, allowing room in the bag for the sample volume to increase 1.5-2-fold during dialysis.

b. Dialyze solution at 4°C against 4 liters of dialysis buffer. Change the buffer three timesat intervals of ~6 hours.

Because of the high viscosityof the D Asolution, dialysis generally takes ~24 hours to complete.

To ISOlATE DNA THAT HAS AN AVERAGE SIZE OF 100-150 KB

a. After the third extraction with phenol, transfer the pooled aqueous phases to a fresh centrifuge tube and add 0.2 volume of 10 M ammonium acetate. Add 2 volumes of ethanol at room temperature and swirl the tube until the solution is thoroughly mixed.

b. The DNA immediately forms a precipitate. Remove the precipitate in one piece from the

ethanolic solution with a Shepherd's crook (a Pasteur pipette whose end has been sealed

and shaped into a U; please see Steps 5-7 of Protocol 3). Contaminating oligonucleotides

remain in the ethanolic phase.

c. If the DNA precipitate becomes fragmented, abandon the Shepherd's crook and collect

the precipitate by centrifugation at SOOOg(6500 rpm in a Sorvall SS-34) for 5 minutes at room temperature.

d. Wash the D A precipitate twice with 70% ethanol, and collect the DNA by centrifugation as described in Step c.

e. Remove as much of the 70% ethanol as possible, using an aspirator. Store the pellet of DNA in an open tube at room temperature until the last visible traces of ethanol have

evaporated.

Do not allow the pellet of DNAto dry completely;desiccatedDNAis very difficult to dissolve.

f. Add I ml ofTE (pH 8.0) for each 0.1 ml of cells (Step I). Place the tube on a rocking platform and gently rock the solution for 12-24 hours at 4°C until the DNA has completely dissolved. Store the DNA solution at 4°C.

10. Measure the concentration of the DNA.

It is often difficult to measure the concentration of high-molecular-weight DNA by standard methods such as absorbance at 260 nm. This is because the DNA solution is frequently nonhomogeneous and is usually so viscous that it is impossible to withdraw a representative sample for analysis. These problems can be minimized by withdrawing a large sample (I 0-20 ~I) with an automatic

pipetter equipped with a cut-off yellow tip. The sample is then diluted with -0.5 ml of TE

(pH 8.0) and vortexed vigorously for 1-2 minutes. The absorbance of the diluted sample can then be read at 260, 270, and 280 nm in the standard way.

A solution with an A,60 of 1 contains -50 ~g of DNA/mI. Note that estimates of purity of nucleic acids based on OD,60:0D280 ratios are unreliable (Glasel1995) and that estimates of concentration are inaccurate if the sample contains significant amounts of phenol. H,O saturated with phenol absorbs with a characteristic peak at 270 nm and an OD'60:0D280 ratio of 2 (Stulnig and Amberger 1994). Nucleic acid preparations free of phenol should have OD,60:0D,80 ratios of -1.2. For further information, please see Appendix 8.

More accurate measurement of DNA concentrations can be made by fluorometry in the presence of fluorescent dyes such as SYBR Gold and Hoechst 33258, which bind DNA without intercalating and with specificity to double-stranded DNA.

11. Analyze the quality of the preparation of high-molecular-weight DNA by pulsed-field gel electrophoresis or by electrophoresis through a conventional

0.6% agarose gel. Use unit-length and/or linear concatemers of A

DNA as markers.

Do not be concerned if some of the DNA remains in the well, since DNA molecules >250 kb have

difficulty entering the gel. This problem can usually be solved by embedding the DNA in a small amount of melted agarose (at 55°C) and transferring the molten solution to the well of a preformed agarose gel. The transfer should be done before the gel is submerged in electrophoresis

buffer.

you can find complete description in Sambrook and Russell book