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Key: DNA1, DNA2, DNA3, ... = Individual DNA samples for T-DNA mapping. Add 5µL DNA (1° reaction) in an entire horizontal row (e.g. A) for each individual. AD1, AD2, AD3, ... = Arbitrary Degenerate primers. Add 5µ of the 4X AD primer (1° reaction) to each vertical column as diagram indicates. lightyellow= Left half of plate-Add LB1 primer cocktail. grey=Right half of plates-Add RB1 primer cocktail. 3. Start the 1° Reaction (detailed in Additional Information) program on thermal cycler and press PAUSE, letting the block cool to 4°C. 4. Mix the LB1 and RB1 cocktails according to TAIL Recipe spreadsheet included. NOTE: Add Taq polymerase last. 5. Add 10µL of each cocktail (LB1 and RB1) to appropriate wells according to previous diagram. 6. Place plate in thermal cycler and press PAUSE, again to allow the reaction to proceed. 7. To prepare the 2° reaction, dilute 1° TAIL reaction 200-fold by transferring 1µL PCR products to 199µL ddH2O. (This is most easily achieved through the use of a multi-channel pipette.) 8. Set up 2° reaction plate according to same diagram, except use 4µL diluted DNA. NOTE: As before, keep plate on icethroughout preparation. 9. Add 5µL of the AD primers to the appropriate wells. 10. Start 2°ree; reaction program on thermal cycler and press PAUSE. 11. Add 11µL of border (LB2 or RB2) cocktail to appropriate wells and place plate in thermal cycler. Press PAUSE to allow reaction to proceed. 12. Once the 2° reaction has completed, the products can either be sequenced or a 3° reaction can be run to further purify the PCR products if there are many nonspecific products. CONTINUE if a 3° reaction is needed. To prepare samples for sequencing, SKIP to step 25. 13. The 3° reaction is prepared like the 2° needs to be diluted 100-fold and the overall reaction volume is 50µL. Add the diluted products from the 2° reaction to a new PCR plate. Again, keep reaction on ice and use a multi-channel pipette for diluting. 14. Add 12.5µL of the AD primers to the appropriate wells. 15. Start the 3° reaction program on the thermal cycler and press PAUSE. 16. Mix the LB3 and RB3 cocktail (adding the Taq last) and add 32.5µL to appropriate half of plate. 17. Flash spin in a table top centrifuge to assure all reaction contents are at the bottom of the wells. 18. Place plate in thermal cycler and press PAUSE again to allow reaction to proceed. 19. To sequence entire contents of plate, SKIP to step 25. To run a gel and visualize the 3°ree; reactions, gollow these steps: Prepare a large 1% agarose gel with 4 rows of 50 wells (200 total wells). 20. Add the appropriate ladder (100bp or 1kb) to the first and last well in each row. 21. Using a multi-channel pipette, draw 7µL from row A or the 2° reaction. Expel this amount on a piece of parafilm. Using the same pipette tips, draw 3µL of loading dye and add it to the droplets on the parafilm. Mix the dye and reaction contents by pipetting up and down. 22. Without changing tips, draw all 10µL of the samples and add them to the gel starting next to the ladder in the top, left portion of the gel. NOTE: Using the multi-channel pipette will leave a space between the samples, this isdesired. 23. Discard the pipette tips and repeat previous step until entire 2° reaction contents are loaded into the gel. Assure a space is left between all 2° reactions added to gel. 24. Now, do the same with the 3° reactions, add the 10µL of the 3° reactions directly next to the 2° reactions. If loaded properly, all lanes will be filled without spaces. This will make the gel easier to analyze. There should be a visible shift in product length from the 2° to the 3° raction. If there are multiple bands visible in one lane, purify individual bands for sequencing via the Topo Cloning Procedure. If single bands exist in the 3° reaction, continue to step 25 for product purification. 25. The PCR products must be purified before they can be squenced. This can be done individually via the Qiagen PCR purification protocol or enzyme purified as explained in this protocol. Transfer 5µ: of 2° reaction PCR products to a new plate. (Again, this is very easy with a multi-channel pipette). 26. Mix the Enzyme (Exol/SAP) Purification cocktail as follows:
27. Add 2.0µL enzyme purification cocktail to DNA samples (on ice). Flash spin plate in a tabletop centrifuge. 28. Run reaction in thermal cycler. Use following program: Step 1= 37°C for 20 min. Step 2= 80°C for 15 min. Step 3= 4°C forever Step 4= END **The reactions are now ready to be sequenced with the corresponding border primers. ADDITIONAL INFORMATION TAIL 1° REACTION PROGRAM: Control Method: CALCULATED
1=4° for 2 min.
2=93° for 1 min.
3=95° for 1 min.
4=94° for 30 sec.
5=62° for 1 min.
6=72° for 2 min. 30 sec.
7=Go to step 4 for 4 more cycles
8=94° for 30 sec.
9=25° for 3 min.
10=Ramp for 72° at 0.2°/sec, 72° for 2 min. 30 sec.
11=94° for 10 sec.
12=68° for 1 min.
13=72° for 2 min. 30 sec.
14=94° for 10 sec.
15=68° for 1 min.
16=72° for 2 min. 30 sec.
17=94° for 10 sec.
18=44° for 1 min.
19=72° for 2 min. 30 sec.
20=Go to step 12, for 14 more cycles
21=72° for 5 min.
22=4° forever
23=END
Control Method: CALCULATED
1=4° for 2 min.
2=94° for 10 sec.
3=64° for 1 min.
4=72° for 2 min. 30 sec.
5=94° for 10 sec.
6=64° for 1 min.
7=72° for 2 min. 30 sec.
8=94° for 10 sec.
9=44° for 1 min.
10=72° for 2 min. 30 sec.
11=Go to step 2, for 11 more cycles
12=72° for 5 min.
13=4° forever
14=END
Control Method: CALCULATED
1=4° for 2 min.
2=94° for 10 sec.
3=44° for 1 min.
4=72° for 2 min. 30 sec.
5=Go to step 2, for 19 more cycles
6=72° for 5 min.
7=4° forever
8=END
Stock concentrations of AD primers should be 20µM. To achieve the concentrations required for TAIL reactions, dilute in a seperate tube. The final amount of 400µL is sufficient for all 3 TAIL reactions. 64-fold degenerate Add 160µL primer and 240µL ddH2O 128-fold degenerate Add 240µL primer and 160µL ddH2O 256-fold degenerate Add 320µL primer and 180µL ddH2O Alternate Plate Setups for TAIL If certain AD primers and/or border primers are found to produce more reliable products there is no need to use the other primers. As an example, I found the LB primer to work more often than the RB primer. Similarly, I found the AD1 and AD4 primers to generate nonspecific (vector) products at a high rate. Therefore, I designed a plate using ONLY the LB primer and AD2, AD3, AD5, and AD6 primers. This increased the maximum amount of DNA samples that I could run on one plate from 8 to 24. Obviously this can save a lot of time and materials. Here is an example of the modified plate setup.
** Note that only left border is used in the entire plate If an alternate setup is used, remember to modify the cocktail for each reaction via the TAIL Recipe setup sheet. The recipe can be manipulated to accommaodate any number of AD primers and individual DNA samples. If the cocktail volume is greater than 1.5mL, two tubes will be needed to prepare the cocktail; divide each components' value by two and use 2 tubes. |