Option (A) Isolation of DNA by NaOH
50 mM NaOH
Add 1 g of NaOH to 500 ml of sterile distilled H2O. Can be stored at room temperature for years.
1M Tris, pH 8.0
Add 12.11 g of Tris to approx. 60 ml of sterile distilled H2O, adjust pH to 8.0 with HCl, adjust volume to 100 ml. Can be stored at room temperature for years.
Option (B) DNA isolation using TRI reagent
8 mM NaOH
Add 0.16 g of NaOH to 500 ml of sterile distilled H2O. Can be stored at room temperature for years.
DNA washing solution
100 mM sodium citrate tribasic hydrate in 10% (vol/vol) ethanol. Usually mix 13 g of sodium citrate tribasic hydrate with 500 ml of 10% ethanol.
Option (C) Isolation of DNA by proteinase K
Comprises 100 mM Tris, 5 mM EDTA-disodium (pH 8.0), 0.2% SDS and 200 mM NaCl. For preparation of 1 liter of lysis buffer, mix 10 ml 20% (wt/vol) SDS, 12.11 g Tris, 10 ml 500 mM EDTA (pH 8.0) and 11.68 g NaCl in a small volume of distilled water and adjust to the final volume of 1 liter with distilled water. To prepare 500 mM EDTA, add 186.15 g of EDTA to 500–700 ml of distilled water, mixing and gradually adding NaOH granules until EDTA is dissolved completely. Adjust pH to 8.0 with HCl; adjust volume to 1 liter with distilled water. Solution can be stored at room temperature for up to 1 year.
Prepare 500 μl aliquots with Proteinase K concentration 20 mg/ml. Can be stored at -20°C for several years.
5x PCR buffer total for REDTaq® DNA Polymerase
5x PCR buffer total for REDTaq® DNA Polymerase should contain 50 mM Tris-HCl (pH 8.3), 250 mM KCl, 7.5 mM MgCl2 CRITICAL The concentration of MgCl2 must be exact, 1 mM dNTP, and 0,05% Gelatin.
For 50 ml of 5x PCR buffer total mix 25 ml of 10x REDTaq® PCR Reaction Buffer, 500 μl of each 100 mM dNTPs (dATP, dCTP, dGTP, dTTP), 2 ml of 50 mM MgCl2 (CRITICAL Optimal concentration of MgCl2 for certain primers might differ) and 21 ml sterile distilled H2O. Make 750 μl aliquots. Can be stored at -20°C for up to 2 years. To prepare 50 mM MgCl2 add 0.24 g to 50 ml of sterile distilled water.
Use 10x REDTaq® PCR Reaction Buffer supplied with polymerase or prepare yourself. 10x PCR buffer contains 100 mM Tris-HCl (pH 8.3, SERVA Electrophoresis, cat. no. 37190, Sigma-Aldrich, cat. no. H1758), 500 mM KCl (Sigma-Aldrich, cat. no. P9333), 11 mM MgCl2 and 0.1% gelatin (Sigma-Aldrich, cat. no. G9391), can be stored at -20°C for years.
Research Genetics primers has suitable concentration 6.6 μM. Generi Biotech sells lyophilized primers, which are dissolved to concentration 0.1 mM and they have to be diluted to 6.6 μM. Usually 13.2 μl of primer solution is added to 186.8 μl of sterile distilled H2O. Can be stored at -20°C for years.
0.5x TBE contains 44.6 mM Tris, 44.5 mM boric acid and 1 mM EDTA (pH = 8.0).
Prepare stock solution of 10x TBE. For 1 l mix 108 g of Tris, 55 g of boric acid and 40 ml of 500 mM EDTA. Undissolved white clumps may be made to dissolve by placing the bottle of solution in a hot water bath. Can be stored at room temperature for at least year.
To prepare 500 mM EDTA, add 186.15 g of EDTA to 500–700 ml of distilled water, mixing and gradually adding NaOH granules until EDTA is dissolved completely. Adjust pH to 8.0 with HCl; adjust volume to 1 liter with distilled water. Solution can be stored at room temperature for up to 1 year.
For 0.5x TBE dilute 10x TBE 20 times. Can be stored at room temperature for at least year.
Loading buffer should contain 1% of orange G in 50% (vol/vol) glycerol. Can be stored at 4°C for years. Usually add 1.13 g of orange G to 100 ml 50% (vol/vol) glycerol.
50 bp standard
50 bp standard should contain 83.3 mg/ml 50 bp DNA ladder in 58% (vol/vol) loading buffer. For 600 μl mix 50 μl of 1 g/ml 50 bp DNA ladder, 200 μl of H2O and 350 μl of loading buffer. Store as 100 μl aliquotes at -20°C (can be stored for years). Store currently used aliquote at 4°C (can be used for several months).
Stock solution should contain 1 mg/ml of Ethidium bromide (EtBr) in H2O. Mix solution until all EtBr is dissolved (use vortex or magnetic mixer). ! CAUTION Work with EtBr powder in fume hood, prevent inhalation. Store in the dark (cover vial with aluminium foil). Can be stored at room temperature for years.
A. DNA preparation
Prepare DNA from mouse tails or from tissues. DNA can be prepared in one of three alternative ways: Option A: Extraction by NaOH (suitable for majority of markers). In some cases you might need better quality of DNA then use more laborious Option B: DNA isolation using TRI reagent or Option C: Proteinase K method.
A: Isolation of DNA by NaOH (based on Truett, et al. (29))
1st step TIMING ~1 min per sample + 90 min for incubation
(i) Add 600 μl of 50 mM NaOH to 1.5 ml microtube with 2 mm piece of mouse tail.
(ii) Let the tail in NaOH in 90°C for 90 min (water bath with shaking).
2nd step TIMING ~1 min per sample + 60 min for centrifugation
(i) For neutralization add 50 μl of 1M Tris pH 8.0 and vortex the samples.
(ii) Centrifuge tubes for 60 min at 3220 g at 4°C. Pour supernatant to new tube (0.5 ml). PAUSE POINT DNA can can be stored in a freezer at -20 or -70 °C for years.
(iii) Concentration of DNA is approximately 40 ng/μl. For PCR dissolve DNA 1:10 in sterile distilled H2O. If you need precise concentration of DNA measure concentration using the NanoDrop spectrophotometer.
B: DNA isolation using TRI reagent TIMING ~4 h
(i) Homogenize 4 mm of the mouse tail or 50–100 mg of the tissue sample (fresh or frozen) with 1 ml of TRI reagent in a microtube using a Polytron homogenizer. CRITICAL Sample volume should not exceed 10% of the volume of TRI reagent used for homogenization. Leave the homogenate for 5 min at room temperature (21–23 °C).
(ii) Add 0.2 ml of chloroform per 1 ml of TRI reagent and mix vigorously. Leave the resulting mixture for 2–15 min at room temperature and centrifuge at 12,000 g for 15 min at 4 °C.
(iii) Remove the aqueous phase overlying the interphase.
(iv) Precipitate DNA from the interphase and organic phase with 0.3 ml of 96% ethanol (vol/vol) per 1 ml of TRI reagent used for homogenization; thereafter, mix samples by inversion. Leave the samples at room temperature for 2–3 min and centrifuge at 2,000 g for 5 min at 4 °C.
(v) Remove the supernatant.
(vi) Wash the pellet twice in 1 ml of DNA washing solution. At each wash, leave the DNA pellet in the DNA washing solution for 30 min at room temperature with periodic mixing by hand and centrifuge at 2,000 g for 5 min at 4 °C; discard the supernatant.
(vii) Suspend the DNA pellet in 1 ml of 75% ethanol (vol/vol). Set aside for 10–20 min at room temperature with periodic mixing by hand and centrifuge at 2,000 g for 5 min at 4 °C.
(viii) Remove ethanol and briefly air-dry DNA pellets by keeping tubes open for 5 min at room temperature.
(ix) Dissolve DNA pellets in 0.3 ml of 8 mM NaOH by slowly passing through the pipette tip. Leave DNA samples for about 1 h at room temperature to dissolve.
(x) Centrifuge at 12,000 g for 10 min to remove insoluble material and transfer the resulting supernatant containing DNA to new tubes.
(xi) Measure DNA concentration using a NanoDrop spectrophotometer. PAUSE POINT DNA can be left overnight at 4 °C or stored in a freezer at -20 or -70°C for years.
(xii) For PCR dilute DNA in sterile distilled water to concentration 4 ng μl−1.
C: Isolation of DNA by proteinase K Proteinase procedure TIMING ~3 days
(i) Add 750 μl of lysis buffer, containing 100 μg ml−1 of proteinase K (add 5 μl of 20 mg/ml solution per 1 ml of lysis buffer) to 4 mm of the mouse tail. Lyse the samples at 55 °C overnight.
(ii) Centrifuge the samples for 60 min at 3,220 g at 4 °C to obtain a firm pellet.
(iii) Transmit the supernatant carefully to the microtube with isopropanol (1:1) for precipitation.
(iv) Using disposable inoculation loop withdraw precipitated DNA and transfer it into 0.5 ml of sterile distilled water. Leave DNA samples overnight at 4 °C to dissolve. PAUSE POINT DNA can be stored in a freezer at -20 or -70 °C for years.
(vi) For PCR dilute DNA 1:10 in sterile distilled water to approx. concentration 4 ng μl−1. If you need precise concentration of DNA measure concentration using the NanoDrop spectrophotometer.
20 μl of reaction mix should contain 0.11 µM forward and reverse primers, 0.2 mM concentration of each dNTP, 1.5 mM MgCl2 (CRITICAL Optimal concentration of MgCl2 for certain primers might differ), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 0.4 U of REDTaq® DNA Polymerase and approximately 40 ng of sample DNA.
1st step TIMING: ~15 min
Prepare PCR master mix. For different numbers of reactions see Table 1. Vortex and centrifuge PCR master mix CRITICAL Add Taq polymerase immediately before adding mix to samples.
2nd step TIMING: ~30 s per sample
(i) To each well of PCR plate put 10 μl of DNA sample (aproxx. 4 ng/μl) and add 10 μl of PCR master mix. In electrophoresis gel is distance between wells twice longer then in 96-well plate. For easier application of samples on a gel (if multichannel pipette is used) use the arrangement shown in Table 2.
(ii) Close wells by sealing tape or caps to prevent evaporation. Centrifuge the plate to flow down all reagents on the bottom of the wells.
3rd step TIMING: ~2 h 45 min
Perform PCR reaction using the DNA Engine Dyad® Peltier Thermal Cycler (Bio-Rad, Hercules, CA) or similar device, according to the following scheme: an initial hot start 3 min at 94°C, followed by 40 cycles of 94°C for 30 s for denaturing, 55°C for 60 s for annealing (CRITICAL optimal annealing temperature for certain primers might differ), 72°C for 60 s for elongation, and finally 3 min at 72°C for final extension. Use heated lid during PCR (100°C). PAUSE POINT PCR product can be stored at -20°C for years.
1st step TIMING ~2.5 h
(i) Prepare 3% agarose gel in electrophoresis plastic tray (CRITICAL optimal gel concentration differ depending to size of PCR products analyzed: larger DNA fragments require a gel with larger pores (lower agarose percentage); smaller DNA fragments require a gel with smaller pores (higher agarose percentage)). For 3% agarose gel size 23.8 x 7.5 cm boil in microwave oven 3 g Methaphore agarose and 0.75 g UltraPure™ Agarose (this agarose is added for better mechanical properties of the gel) in 125 ml of 0.5x TBE buffer until it melts. CRITICAL For good dispersing add agarose to buffer, DO NOT pour buffer on agarose powder; optimal gel size may differ for certain primers. Add 7 μl of EtBr from the stock solution (1 mg/ml).
(ii) After approx. 10 min in room temperature pour gel on tray and insert comb. Let in room temperature for 1 hour. PAUSE POINT Can be stored at 4°C in plastic bag for approx. one month.
(iii) Put tray into electrophoresis chamber and overlay it with 0.5x TBE buffer. Add 75 μl of EtBr stock solution (1 mg/ml), put approximately two-thirds of EtBr solution near to anode and the rest near to cathode (for electrophoresis with approximately 1.5 l of buffer).
2nd step TIMING Depends on product length and product length difference (30 min - 3 hours).
(i) Add 1 μl of loading buffer per 4 μl of PCR product solution (in Sigma REDTaq® DNA Polymerase 1U/μl solution is already enough of glycerol as well as dye and loading buffer is not necessary).
(ii) Put 10 μl of each sample solution to starting well in agarose electrophoresis (use multichannel pipette). 3 μl of standard 50 bp DNA should be loaded in the first well.
(iii) Electrophorese samples at constant voltage 150 V (CRITICAL optimal voltage may differ for certain primers), check after approximately 10 min the distance of samples from the start using Bio imaging system (Syngene) or similar device. According product size and length difference different time is needed to see the results. Usually for products length up to 100 bp it is 15-30 min, for length 100-200 bp it is 30-60 min and for length over 200 it is 45-120 min. Make photo of your result by Bio imaging system (Syngene) or similar device. CRITICAL Make photo immediately after taking gel from electrophoresis apparatus to prevent blurring.