TAIL-PCR Protocol
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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
AD1 |
DNA1
AD2 |
DNA1
AD3 |
DNA1
AD4 |
DNA 1
AD5 |
DNA1
AD6 |
DNA1
AD1 |
DNA1
AD2 |
DNA1
AD3 |
DNA1
AD4 |
DNA1
AD5 |
DNA1
AD6 |
DNA2
AD1 |
DNA2
AD2 |
DNA2
AD3 |
DNA2
AD4 |
DNA 2
AD5 |
DNA2
AD6 |
DNA2
AD1 |
DNA2
AD2 |
DNA2
AD3 |
DNA2
AD4 |
DNA2
AD5 |
DNA2
AD6 |
DNA3
AD1 |
DNA3
AD2 |
DNA3
AD3 |
DNA3
AD4 |
DNA 3
AD5 |
DNA3
AD6 |
DNA3
AD1 |
DNA3
AD2 |
DNA3
AD3 |
DNA3
AD4 |
DNA3
AD5 |
DNA3
AD6 |
DNA4
AD1 |
DNA4
AD2 |
DNA4
AD3 |
DNA4
AD4 |
DNA 4
AD5 |
DNA4
AD6 |
DNA4
AD1 |
DNA4
AD2 |
DNA4
AD3 |
DNA4
AD4 |
DNA4
AD5 |
DNA4
AD6 |
DNA5
AD1 |
DNA5
AD2 |
DNA5
AD3 |
DNA5
AD4 |
DNA 5
AD5 |
DNA5
AD6 |
DNA5
AD1 |
DNA5
AD2 |
DNA5
AD3 |
DNA5
AD4 |
DNA5
AD5 |
DNA5
AD6 |
DNA6
AD1 |
DNA6
AD2 |
DNA6
AD3 |
DNA6
AD4 |
DNA 6
AD5 |
DNA6
AD6 |
DNA6
AD1 |
DNA6
AD2 |
DNA6
AD3 |
DNA6
AD4 |
DNA6
AD5 |
DNA6
AD6 |
DNA7
AD1 |
DNA7
AD2 |
DNA7
AD3 |
DNA7
AD4 |
DNA 7
AD5 |
DNA7
AD6 |
DNA7
AD1 |
DNA7
AD2 |
DNA7
AD3 |
DNA7
AD4 |
DNA7
AD5 |
DNA7
AD6 |
DNA8
AD1 |
DNA8
AD2 |
DNA8
AD3 |
DNA8
AD4 |
DNA 8
AD5 |
DNA8
AD6 |
DNA8
AD1 |
DNA8
AD2 |
DNA8
AD3 |
DNA8
AD4 |
DNA8
AD5 |
DNA8
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.