Протоколы выделения ДНК для ПЦР

Выделение ДНК из растительного материала для проведения ПЦР (поиск ГМ-вставок)

Выделение ДНК из сои (Protocols for working with Phytoplasmas)

These are the methods we use to work with Phytoplasmas in lab http://www.plantpath.wisc.edu/

Hot CTAB DNA extraction for soybean phytoplasma analysis
(modified from Zhang et. al. 1998. J. Virological Methods. 71: 45-50)
(Mary E. Lee - 8-31-98)

Remember: Turn on the waterbath at least 2-3 hours before use, fill with distilled water only. Put your bottle of CTAB buffer in also to warm up well before you need it! Also, spray down the countertop with windex and wipe clean before beginning extractions to prevent contamination.

1. Grind 0.3g of the YOUNGEST, actively growing soybean tissue (midveins) in a bleached (20% clorox overnight), autoclaved mortar and pestle with liquid nitrogen until a fine powder is obtained. Change gloves between every sample to prevent contamination.
   
2. Transfer the powdered tissue to a 1.5 mL microcentrifuge tube using a bleached, autoclaved utensil (rubber policeman, spatula).
   
3. Add 800 uL of CTAB buffer, 60C. Remember to add 20 uL mercaptoethanol per 10mL of CTAB buffer before use. (CTAB buffer = 2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 0.2% mercaptoethanol).
   
4. Incubate 20 minutes at 60C in water bath (these can be done in batches of five at a time).
   
5. Place tubes on ice.
   
6. Add 600 uL of chloroform : octanol (24:1) In the hood. Aliquot this to a bleached, autoclaved beaker before using so you don’t contaminate the bottle.
   
7. Vortex vigorously, then vortex again.
8. Centrifuge 6 minutes 14,000 rpm.
   
9. Draw off supernatant and place in a clean microfuge tube. Do this in the hood, and dispose of the chloroform appropriately. Spin again for 2 min 14,000rpm and draw off supernatant again to a new microfuge tube to get rid of any residual plant material.
   
10. Add 600 uL of ice-cold isopropanol (kept in –20C). Aliquot isopropanol to a small bleached autoclaved beaker before use.
    
11. Immediately centrifuge 8 minutes, 14,000 rpm, room temperature.
12. Decant supernatant into a large beaker, wash pellet with approximately 100-200 uL of ice-cold 75% Ethanol made from absolute ethanol and sterile distilled water (stored at –20C). This should also be aliquoted to a bleached, autoclaved beaker before use.
    
13. Let air dry or aspirate off remnant ethanol with plugged pipet tip.
    
14. Resuspend pellet in 100 uL TE using plugged tips. Leave overnight in fridge or in freezer for long-term storage.

Выделение ДНК из растительных тканей (CTAB DNA extraction from plant tissues)

Fill a 2ml Eppendorf tube with about 500µl of glass beads (diameter = 1mm). Place 1 small leave in the tube (1-2 cm²). Snap freeze with liquid N₂ and freeze-dry the samples (or store them at -80°C until freeze-dryer is available).

1. Prepare extraction buffer EB and place it at 65°C.
2. Grind the leaves to a fine powder for 10 seconds using a vigorous shaker (3°C room).
3. Add 700µl of the preheated extraction buffer EB, shake vigorously and immediately place back at 65°C.
4. Incubate at 65°C for 1, 2 or more hrs (longer incubations result in cleaner pellets).
5. Extract with an equal volume of Chloroform:Isoamylalcol, shake for 5 min and centrifuge the samples for 10 min at room temperature at 14000 rpm.
6. Transfer the upper phase (containing the DNA) to a new 1.5ml tube. Precipitate the DNA with 2/3 volume of isopropanol(5min at RT).
7. Spin down the precipitated nucleic acids by centrifuging at R.T. for 10 min at 14000rpm.
8. Discard the supernatant and wash the pellet twice with Wash Buffer WB
9. Air-dry the pellet and re-suspend in 300µl of TE.
10. Add 3µl of RNAse (10mg/mL) and incubate 30 min at 37°C.
11. Bring the dissolved nucleic acid to 2M NaCl (=add 200µl of 5M NaCl).
12. Re-precipitate by adding 2 volumes of 100% Ethanol. Incubate at -20°C for 20 min.
13. Wash the pellet with 500µl 70% ethanol, air-dry the pellets and re-suspend in sterile water or TE.

EB CTAB Extraction Buffer