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
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 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.
1. All reagents and reagent stocks are made with sterile HPLC quality water and are only used for PCR.
2. Plan your experiments to fit the format of your plates or tubes (eg 96-well plates) and to maximize the use of multi-channel pipettors. This minimizes the amount of time reagents are in the wells before PCR cycling begins, thus minimizing mis-primed products.
3. Use reagent boats or strip tubes for holding reagents while reactions are set up. This will allow you to use a multi-channel pipettor for most additions, thus speeding up the process. You can use pipette tip box lids to cover the reservoirs to prevent passive contamination.
4. Taq DNA Polymerase (we use Invitrogen Life Technologies #18038) should be protected from temperature fluctuations by storing and transporting in -20°C cooler boxes.
5. We use Invitrogen's pre-mixed 10 mM dNTPs (#18427-013) as a 50x stock in the TAIL reactions. If you are not doing 96-well format reactions, sub-aliquot the solution so that the nucleotides do not go through more than one freeze/thaw cycle.
6. The primers are sensitive to freeze-thaw cycles and temperature fluctuations. Sub-aliquot to single-plate amounts and store in boxes that insulate them from freezer temperature fluctuations. Include ~15% excess volume. They are stable on ice for the day, so aliquots thawed for TAIL1 may be used again for TAIL2.
Use freshly unwrapped pipette tips and a carefully stored dedicated TE8 solution for primer stock preparation. Keep everything on ice. Dissolve lyophilized primers in TE8 to a concentration of 100 pmol/ml. Make dilutions in TE8, then aliquot in amounts that are typically needed in a day. For primers in regular use, aliquot the entire amount to avoid multiple freeze/thaw cycles. We aliquot our primer sets to PCR strip-tubes for easy pipetting. Store at -20°C.
Mu TAIL PCR reaction: TAIL 1
1. Add 2 ml of the diluted template DNAs to the appropriate wells in a plate kept on ice. Be sure to include a no template sample for each AP. Note that primer GAG3 self-anneals, so you generally will see small bands in this control lane on a gel. All other control lanes should be blank.
2. Add 2ml of AP to each well. 3. Turn on the thermal cycler and have it pause at the initial 95°C step to preheat the block and lid. 4. Calculate the number of reactions you have ([#templates x #primers x #replicates] + #primers). Add about 15% to get the number of reactions to prepare in your Master Mix (MM). Keep the MM on ice.
5. Put the MM into a reagent boat and add 16ml to each well. Seal plate or tubes, vortex gently. Briefly spin down in a refrigerated tabletop centrifuge. Place in thermal cycler and initiate the TAIL1 program.
6. TAIL2 is initiated immediately after TAIL1 is completed. The TAIL1 reactions may be stored for a short period of time at 4°C. At this writing, I do not know how long they can be stored and still generate great TAIL2 products. I have not had good luck doing the TAIL2 reactions the next day. Storing at -20°C degrades the quality of the TAIL1 products for TAIL2.
7. Check 10 ml aliquots of each TAIL1 reaction by gel electrophoresis in a 1% TBE gel. In the gel photo above, our size marker is 1 mg of the Invitrogen 1kb DNA ladder. The TAIL products should range in size from ~200-4000 bp with the bulk of them being in the mid-range. As an example, the photo above shows products from two DNA templates primed with 10 different primers. The blank lanes are no template controls. Primer #8 reactions failed, as did primers #2 and #6 with template A. The faint band at the top of the lane is template DNA. geeky1 reactions usually appear smeared—this is ok.
Mu TAIL PCR reaction: TAIL 2