| Thermal Asymmetric Interlaced Tail PCR | 点击： 作者：51protocol收集 来源： 时间： 2007-03-29 本站论坛
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|  | Purpose: Mu TAIL PCR produces a population of fragments with one end anchored in Robertson's Mutator (Mu) TIR's and the other in the Mu flanking DNA.  These products may be used for cloning, gel analysis, or as hybridization probes. This protocol details the steps to take to improve the chances for uncontaminated and reproducible TAIL reactions. The protocol is adapted from the published method (Liu, Mitsukawa, Oosumi, and Whittier 1995. The Plant Journal. 8(3):457-463).Template preparation: Fast Isolation of Genomic DNA This protocol is used for quick isolation of genomic DNA from plant materials. Other methods may also prove effective. DNA extraction buffer: (For 400 ml solution) Store at RT. 168 g Urea
25 ml 5 M NaCl
20 ml 1 M Tris-HCl pH 8.0
16 ml 0.5 M EDTA
20 ml 20% Sarkosyl
190 ml distilled H2O 1. Grind about 1 g of fresh seedling leaves, or other tissues, to a fine powder in liquid nitrogen using a mortar and pestle. Use more tissue from other plant parts or older plants. Be careful to avoid contaminating adjacent samples with the powder.2. Add 5 ml DNA extraction buffer. Mix gently with the pestle to evenly distribute the powdered tissue in the buffer.3. Let it thaw to room temperature and mix gently with the pestle again.4. Transfer to a polypropylene centrifuge tube (eg Falcon 352006).5. Add 4 ml TE8-(10mMTris-HCl, 1mM EDTA, pH8)-saturated phenol:chloroform (1:1) (bottom phase). Close cap. Shake briefly with hands and put the tubes, laying horizontally in a tightly closed container, on the belly dancer or similar gyrating platform mixer at the highest speed for at least 30 min. Wear gloves and goggles when handling phenol:chloroform.6. Spin at ~1625g for 5-20 mins (=max speed on an IEC CL centrifuge)(max speed in 1222 ‘fuge). There will be a solid middle phase that separates the organic bottom layer from the aqueous top layer.7. Pour the upper layer into a fresh centrifuge tube (15 ml screw-cap conical). You may have to use a pipette if the middle pellet is not thick enough. The aqueous volume should be about 5 ml. (The phenol:chloroform phase is transferred to a designated waste container.)8. To the supernatant, add 0.5 ml 3 M NaOAc (pH 5.2). Mix gently by inverting the capped tube several times and a color change will occur.9. Add 3.9 ml isopropanol. Mix gently by inversion. In most cases, a clump of thread-like DNA is visible.10. Either use a glass pipette hook to fish out the DNA if it is visible (preferred), or centrifuge at 10,000 rpm for 10 min to pellet the DNA.11. Wash the DNA clump by transferring it to a microcentrifuge tube containing 1 ml 70% EtOH. Centrifuge at 10,000 rpm for 5 min at 4°C.12. Drain the EtOH by carefully inverting the tube on a KimWipe for a few minutes, then dry the DNA in a vacuum dryer for about 10 min. Do not overdry as it will become nearly insoluble.13. Add 200 – 400 ml TE8 buffer to dissolve the DNA. Leave it at 4 °C a few hours or overnight. The concentration should be approximately 100 ng/μl. Run 1 μl with and without DNase-free RNase on a 0.8% gel to check the prep for quality and concentration.14. To minimize freeze-thaw cycles, make 3 μl aliquots of each DNA in PCR tubes and store at -20°C. Multiple DNA samples can be arrayed in strip tubes for convenient dispensing into the reaction later. Use the aliquots within a few months.15. To use, make a 1:20 dilution by adding 57 μl of HPLC quality water to each aliquot. In some cases, this dilution should be adjusted if the DNA prep is at a concentration much different from 0.1mg/ml. Keep on ice. Discard after one use—do not store the dilution nor refreeze the aliquot. Lab bench hygiene: contamination prevention, done for each experiment 1. Wash the lab bench with a mixture of bleach, detergent and water. 2. Wipe down freezer and refrigerator handles with dilute bleach. 3. Wash the boxes you will use to hold the PCR plates and tubes with dilute bleach. 4. Clean your pipettors with 70% EtOH and compressed air (don't lose the gaskets!). It is best to have a set dedicated to PCR that are always used with filter tips. Wipe them down again after pipetting template solutions.5. Never touch anything that you use for TAIL reactions unless you have clean gloves on. Store all disposables in a dedicated drawer that you open only with clean gloves on. 6. Keep lids closed on pipette tip boxes and reagents. Do not use PCR reagents for other purpos 7. Use aerosol-resistant tips for all pipetting steps. 8. Keep everything at 4°C and covered while reactions are set up. I recommend using Eppendorf PCR Coolers which stay at this temperature for 1 hour. As an alternative, some types of pipette tip boxes can be used as ice chests to conveniently hold PCR tubes and plates. 9. Change gloves whenever you leave your bench area or if you handle a DNA solution. Discarded gloves may be re-used for other activities for which contamination is not an issue.
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