Big Dye Primer Sequencing Reaction Protocol
This protocol outlines the three steps involved in sequencing a PCR reaction:
- The preparation of pre-made sequencing plates
- The dilution of PCR products in preparation for sequencing
- The setup of sequencing reactions
In general, we use primer sequencing for all first pass sequencing attempts. Terminator sequencing is used when first pass sequencing reveals an indel. (See Polymerase Chain Reaction Protocol.)
Our experiments have shown that the best way to prepare out PCR samples for sequencing is to dilute them with water.
Our PCR reaction (see Polymerase Chain Reaction protocol) does not call for a huge excess of primers or dNTPs. Instead of removing the excess primers and dNTPS or breaking them down enzymatically, we have found that diluting with water is cheaper, easier and faster.
DNA Engine Tetrad (MJ Research PTC-225)
Jouan Centrifuge (CR422)
Multimek 96 Automated 96-Channel Pipettor (Beckman Instruments)
384-split-well plate (Cat. No. 1055-40-0 from Robbins Scientific)
0.01% Triton H2O
Biosystems Big Dye Primer Forward 5000-reaction kit (Cat. No. 403049)
Applied Biosystems Big Dye Primer Reverse 5000-reaction kit (Cat. No. 403050)
Preparing Pre-Made Sequencing Plates
- Thaw the tubes of Big Dye Primer forward and reverse sequencing reactions. Thaw these in a drawer, away from light.
- Each of these tubes will be aliquoted into two MJ Research PCR plates. Prepare these plates by labeling them with the title, "Big Dye Primer," the specific base (A, C, G, or T), the direction (forward or reverse), and the date.
- Once the reactions have been thawed, vortex briefly and place them on ice.
- To aliquote the reactions, use the Eppendorf Repeater pipette. Dispense 100 uL into each well of the MJ Research plate, keeping this plate in an ice bucket as this is done.
- Cover the completed plates with foil and place in the freezer.
- Any remaining reactions can be placed in an Eppendorf 1 mL tube, labeled, and stored in the freezer.
For the Forward Big Dye Primer Pre-Made Sequencing Plates
- In the upper left-hand corner of 24 384-split-well plates, write "FOR" for the forward reactions. This helps to provide an easy to see orientation for the plate. On 24 foil covers, write "BDP Forward" and the date.
- Fill three buckets with ice.
Note: The number of reactions remaining in the plate will be listed on the foil - this corresponds to the number of sequencing plates that can be made. An unused reaction plate will contain 48 reactions.
- Reset and initialize the Multimet 96-channel pipettor and choose the program 384-Pipetting-2µ-1st quadrant.
- Put a box of tips on the robot deck, along with a thawed plate of BDP Forward A reactions, and three empty 384-well plates (the plates should be sitting in black bases). Start the program. The robot will pick up the tips, aspirate 4 uL of the reaction and add it to the first quadrant of the 384-well plate (labeled "a" on the plate).
- When finished, put the plates on ice and cover with a paper card. Continue in this manner until all the plates have the A Big Dye Primer added.
- Next, work on the "C" reactions. Choose 384-Pipetting-2µ-2nd quadrant. This program adds 4 µL of the C Big Dye Primer to the "b" quadrant of the 384 well plate. Repeat as above, keeping the reactions on ice before and after the C reaction is added.
- Repeat for the "G" and "T" reactions. For "G", choose 384-Pipetting-2µ-3rd quadrant, which adds 2 µL to the "d" quadrant on the plate. The program 384-pipetting-2µL-4th quadrant adds 2 µL of "T" to the "c" quadrant.
- When all of the reations have been added, spin the plates down on the Jouan using the quick spin. Cover with the labeled foil, using the rollers to secures the foil to the plate. Store in the freezer until ready to use.
For the Reverse Big Dye Primer Pre-Made Sequencing Plates:
- Repeat as outlined above, labeling the plates "REV" in the upper left hand corner and labeling the foil covers with "BDP Reverse" and the date.
- The robot programs are exactly the same; the only difference is that instead of "A", "C", "G" or "T" Forward, use "A", "C", "G", or "T" Reverse. These plates will have left-over reagents in them; combine these leftovers, put them in tubes, and store in the freezer.
Dilution of PCR products in preparation for sequencing
- To prepare out PCRs for sequencing, we add 4 uL of water for every microliter of PCR product. We have found that for our method of sequencing reaction setup (see below) it works best to use 0.01% Trition water. Triton water has less surface tension than water alone, and this helps in pipetting small amounts.
Note: Use a smaller dilution (e.g., 2µL or 3µL of water for every microliter of PCR product) if the product produces fainter bonds when run on a gel.
- When adding Triton water, keep in mind that not every column of the PCR plate will have the same amount of PCR product. To check the PCR, we remove 2 uL for the first and last column. Before adding the triton water, it is helpful to spot check the volumes from the first column, the last column and one of the middle columns.
Sequencing Reaction Setup
- To sequence one 96-well plate of PCR reactions, remove one forward and one reverse pre-made sequencing plate.
Note: Even though these plates are spun down before they are stored in the freezer, small bits of frozen reagent usually collect inside the tops of the wells where the foil is placed. It is helpful to melt these frozen bits by running one's hand over the foil and then spinning the plate down before removing the foil.
- Store these plates on ice until the next step (see below) is completed. Preheat two Tetrad alpha blocks.
- Initialize the robot and open the program called Insert. This program mixes the PCR product 25 times (to mix the PCR product thoroughly with the triton water). When the mixing the robot pauses, take the sequencing plates off the ice and place them on the robot deck.
- Start the program again. The robot will add 1 L of the diluted PCR product to each of the four quadrants on the forward plate. This process will be repeated for the reverse plate.
- 6. After the PCR product has been added to the pre-made sequencing plates, spin them down in the Jouan and cover them with Cycle Seal PCR Plate Sealer. Put the reactions in the Tetrad, set the reaction volume to 12 µL, and run the following program:
| ||1. 96° C for 10 sec|
| ||2. 55° C for 5 sec|
| ||3. 70° C for 1 min|
| ||4. Repeat steps 1-3 14 more times|
| ||5. 96° C for 10 sec|
| ||6. 70° C for 1 min|
| ||7. Repeat 4-5 14 more times|
| ||8. Go to 4° C|
When the reaction is done (it takes about 1.5 hours) remove it from the Tetrad and store it in the fridge until the precipitation can be started (see Ethanol Precipitation Protocol).
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