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中生网 > 实验技术 > PCR技术 > TAIL PCR Protocol

TAIL PCR Protocol

更新:2013年10月10日 阅读次数: 【字体:

 

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
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 wellsaccording to previous diagram.

6. Place plate in thermal cycler and press PAUSE, again to allow thereaction to proceed.

7. To prepare the 2° reaction, dilute 1° TAIL reaction 200-fold bytransferring 1µL PCR products to 199µL ddH2O. (This is most easilyachieved 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 andplace plate in thermal cycler. Press PAUSE to allow reaction to proceed.

12. Once the 2° reaction has completed, the products can either besequenced or a 3° reaction can be run to further purify the PCR productsif there are many nonspecific products. CONTINUE if a 3° reaction isneeded. To prepare samples for sequencing, SKIP to step 25.

13. The 3° reaction is prepared like the 2° needs to be diluted100-fold and the overall reaction volume is 50µL. Add the dilutedproducts 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 reactioncontents are at the bottom of the wells.

18. Place plate in thermal cycler and press PAUSE again to allowreaction to proceed.

19. To sequence entire contents of plate, SKIP to step 25. To run a geland visualize the 3°ree; reactions, gollow these steps: Prepare a large1% agarose gel with 4 rows of 50 wells (200 total wells).

20. Add the appropriate ladder (100bp or 1kb) to the first and lastwell 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 samepipette tips, draw 3µL of loading dye and add it to the droplets on theparafilm. Mix the dye and reaction contents by pipetting up and down.

22. Without changing tips, draw all 10µL of the samples and add them tothe 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 thesamples, this is desired.

23. Discard the pipette tips and repeat previous step until entire 2°reaction contents are loaded into the gel. Assure a space is leftbetween 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, alllanes will be filled without spaces. This will make the gel easier toanalyze. 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 forproduct purification.

25. The PCR products must be purified before they can be squenced. Thiscan be done individually via the Qiagen PCR purification protocol orenzyme purified as explained in this protocol. Transfer 5µ: of 2°reaction PCR products to a new plate. (Again, this is very easy with amulti-channel pipette).

26. Mix the Enzyme (Exol/SAP) Purification cocktail as follows:

ddH2O
ExoI
SAP
(PCR Products)
TOTAL

1.4µL
0.2µL
0.4µL
---(5.0µL)
2.0µL (7.0µL)


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 correspondingborder 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

TAIL 2° REACTION PROGRAM:

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

TAIL 3° REACTION PROGRAM:

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

AD (Arbitrary Degenerate) Primer Sequences and Concentrations:
 

AD1: NGTCGASWGANAWGAA
AD2: TGWGNAGSANCASAGA
AD3: AGWGNAGWANCAWAGG
AD4: STTGNTASTNCTNTGC
AD5: NTCGASTWTSGWGTT
AD6: WGTGNAGWANCANAGA

128-fold degenerate 12µM
128-fold degenerate 12µM
128-fold degenerate 12µM
256-fold degenerate 16µM
64-fold degenerate 8µM
256-fold degenerate 16µM



Stock concentrations of AD primers should be 20µM. To achieve theconcentrations 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 morereliable products there is no need to use the other primers. As anexample, 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 usingONLY the LB primer and AD2, AD3, AD5, and AD6 primers. This increasedthe maximum amount of DNA samples that I could run on one plate from 8to 24. Obviously this can save a lot of time and materials. Here is anexample of the modified plate setup.

DNA1
AD2
DNA1
AD3
DNA1
AD5
DNA1
AD6
DNA 9
AD2
DNA9
AD3
DNA9
AD5
DNA9
AD6
DNA17
AD2
DNA17
AD3
DNA17
AD5
DNA17
AD6
DNA2
AD2
DNA2
AD3
DNA2
AD5
DNA2
AD6
DNA 10
AD2
DNA10
AD3
DNA10
AD5
DNA10
AD6
DNA18
A D2
DNA18
AD3
DNA18
AD5
DNA18
AD6
DNA3
AD2
DNA3
AD3
DNA3
AD5
DNA3
AD6
DNA 11
AD2
DNA11
AD3
DNA11
AD5
DNA11
AD6
DNA19
A D2
DNA19
AD3
DNA19
AD5
DNA19
AD6
DNA4
AD2
DNA4
AD3
DNA4
AD5
DNA4
AD6
DNA 12
AD2
DNA12
AD3
DNA12
AD5
DNA12
AD6
DNA20
A D2
DNA20
AD3
DNA20
AD5
DNA20
AD6
DNA5
AD2
DNA5
AD3
DNA5
AD5
DNA5
AD6
DNA 13
AD2
DNA13
AD3
DNA13
AD5
DNA13
AD6
DNA21
A D2
DNA21
AD3
DNA21
AD5
DNA21
AD6
DNA6
AD2
DNA6
AD3
DNA6
AD5
DNA6
AD6
DNA 14
AD2
DNA14
AD3
DNA14
AD5
DNA14
AD6
DNA22
A D2
DNA22
AD3
DNA22
AD5
DNA22
AD6
DNA7
AD2
DNA7
AD3
DNA7
AD5
DNA7
AD6
DNA 15
AD2
DNA15
AD3
DNA15
AD5
DNA15
AD6
DNA23
A D2
DNA23
AD3
DNA23
AD5
DNA23
AD6
DNA8
AD2
DNA8
AD3
DNA8
AD5
DNA8
AD6
DNA 16
AD2
DNA16
AD3
DNA16
AD5
DNA16
AD6
DNA24
A D2
DNA24
AD3
DNA24
AD5
DNA24
AD6


** Note that only left border is used in the entire plate
If an alternate setup is used, remember to modify the cocktail for eachreaction via the TAIL Recipe setup sheet. The recipe can be manipulatedto accommaodate any number of AD primers and individual DNA samples. Ifthe cocktail volume is greater than 1.5mL, two tubes will be needed toprepare the cocktail; divide each components'' value by two and use 2tubes.

 

关键词:PCR Protocol
相关栏目:实验技术 PCR技术
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