TAIL

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TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA lines that are to be mapped. AD primers are degenerate primers that anneal throughout the genome. The border primers are specific for the left and right borders of the T-DNA. From the primary reaction to the tertiary, the border primers get closer to the edge of the T-DNA. That is why a "shift" is visible when running a gel with the secondary and tertiary reactions next to each other. The success rate of TAIL-PCR varies, depending onhow many DNA samples, AD primers, and border primers are used.

1. Dilute the DNA sample 1:5 (Dilute more or less depending on DNA concentration.)

2. Add 5µL DNA, and 5µL AD primers to PCR plate according to the diagram below (each AD primer has a specific concentration, see Additional Information at the end of the protocol):
NOTE: Keep plate on ice throughout the procedure.

DNA1
AD1DNA1
AD2DNA1
AD3DNA1
AD4DNA 1
AD5DNA1
AD6DNA1
AD1DNA1
AD2DNA1
AD3DNA1
AD4DNA1
AD5DNA1
AD6DNA2
AD1DNA2
AD2DNA2
AD3DNA2
AD4DNA 2
AD5DNA2
AD6DNA2
AD1DNA2
AD2DNA2
AD3DNA2
AD4DNA2
AD5DNA2
AD6DNA3
AD1DNA3
AD2DNA3
AD3DNA3
AD4DNA 3
AD5DNA3
AD6DNA3
AD1DNA3
AD2DNA3
AD3DNA3
AD4DNA3
AD5DNA3
AD6DNA4
AD1DNA4
AD2DNA4
AD3DNA4
AD4DNA 4
AD5DNA4
AD6DNA4
AD1DNA4
AD2DNA4
AD3DNA4
AD4DNA4
AD5DNA4
AD6DNA5
AD1DNA5
AD2DNA5
AD3DNA5
AD4DNA 5
AD5DNA5
AD6DNA5
AD1DNA5
AD2DNA5
AD3DNA5
AD4DNA5
AD5DNA5
AD6DNA6
AD1DNA6
AD2DNA6
AD3DNA6
AD4DNA 6
AD5DNA6
AD6DNA6
AD1DNA6
AD2DNA6
AD3DNA6
AD4DNA6
AD5DNA6
AD6DNA7
AD1DNA7
AD2DNA7
AD3DNA7
AD4DNA 7
AD5DNA7
AD6DNA7
AD1DNA7
AD2DNA7
AD3DNA7
AD4DNA7
AD5DNA7
AD6DNA8
AD1DNA8
AD2DNA8
AD3DNA8
AD4DNA 8
AD5DNA8
AD6DNA8
AD1DNA8
AD2DNA8
AD3DNA8
AD4DNA8
AD5DNA8
AD6

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 ice throughout 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.