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:
|
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 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
