Purpose:

Sample is subjected to numerous heating and cooling cycles to denature/re-nature the DNA (separate the DNA strands so sites are available for the primers to attach to and finally analyzed via the 3700)

• Template DNA= Bluescript with insert. During thermal cycling, insert and Bluescript are denatured and than re-natured with the primer bases.

• Check with Marc to be sure that we are signed up to use the thermal cycler downstairs.

Materials:

• Thermal Cycling work Sheets

• 2 Micro Amp 384 Cycle Sequence (CS) Plates
• 2 Micro Amp Clear Adhesive Films for 384 well Reaction Plates (kept under the Hydra, not the blue ones, have tabs on both ends.)
• 2 - ½ strips of aluminum foil, foil is used to cover plates when done

1. Fill out thermal cycling work sheets and record date thermal cycled on summary sheets in respective library notebook.
2. Thaw 4 - 96-well Dynex v-bottom DNA plates (from mini-prep stored in –20C) on Titer Tech Shaker, 6 is a good speed, approximately 1 hour.
3. Label CS plates. 2 - CS plates per 4 samples (one for each primer)
4. Add 2ul water; Hydra Protocol: water addition 2ul to 384 well CS plate 2X.
5. Transfer 2ul of DNA into 384 cycle sequence plates. Hydra Protocol: DNA addition 2ul to 384 well CS plate 2X.
6. While Hydra is preparing to transfer next samples, keep CS plates in 4C refrigerator. This retards evaporation.
7. Immediately proceed with master mix preparation.

Master Mix Materials:

• Blue Cold Box (Stored in –20 chest freezer in the main lab)
• PolyT, JenRev primers and Big Dye (Stored in the EST Box in the –20 freezer)

• 5X Rxn. Buffer (Stored in the 4C refrigerator.
• DMSO
• Tube of sterile water
• Eppendorf Repeat Pipettor
• 2- Small syringe
• 2- 2.0ml screw-cap micro-centrifuge tubes (one for each primer)

Method: (PREPARING 1/12 MASTER MIX REACTION)

• THERE IS NO EXCESS VOLUME IN THIS REACTION, WATCH PIPETTING TECHNIQUE CAREFULLY SO AS NOT TO GET AIR INSTEAD OF MASTER MIX IN THE SYRINGE. KEEP MASTER MIX COLD AND SHIELDED FROM LIGHT.

1. Gather materials.
2. Set up tubes in the cold box. Be sure Primers and Big dye are thawed but cold and covered.
3. Label 1^st tube PT, 2^nd tube JR
4. (107ul X 2 AC ddwater) to each tube, 214ul total based on 4 plates, mark each addition off as it is added.
5. (130ul DMSO) to each tube.
6. (400 + 132ul 5X Buffer) to each tube, 532ul.
7. (56ul Respective Primer) to each tube.
8. (134ul X 2 Big Dye) to each tube, 268ul total.
9. Very carefully vortex and than spin gently to recover volume from the top of the tube.

1. Starting with PT, add 3ul with repeat pipettor (syringe sets dispensing volume, check to be sure it is correct) to each well in PT plate. Seal with adhesive seal, cover with aluminum foil and set in 4C refrigerator until ready to thermal cycle.
2. Prepare JenRev next, add 3ul with repeat pipettor (adapter sets ul) to each well in JR plate. Seal with adhesive seal, cover with aluminum foil and set in 4C refrigerator until ready to thermal cycle.

• Thermal Cyclers are located in the GAF Lab on the 2^nd floor.

• 2 instruments 1 to the right and left of the bench centrifuge. Record on the thermal cycling worksheets which thermal cycler is used 384-right or 384-left, also, note primer location nest 2 or 1.
• Start plates at 4:30 or 5:00 and pickup first thing the next am. G-50 clean up should start immediately after leaving thermal cycler. (Keep plates in 4C refrigerator when they are not in use; remember these plates are light sensitive.)
• Check to be sure 8 G-50 plates are ready for the next day.

1. To thermal cycle, centrifuge both plates up to 1000-1500 RPM and than place on the thermal cycler the GAF lab on the 2^nd floor.
2. Cycle Sequencer Protocol:

• 384 R:

• Power ON
• User PrattLab
• Run CYCLE (without the dots)
• Start 2X (1-heat block rises to correct temp and 2-run)

• 384 L:

• Power ON
• User PrattLab
• Run CYCLE OR THERMAL CYCLE (these are the same)
• Start 2X (1-heat block rises to correct temp and 2-run)

• PROTOCOL PARAMETERS:
• TMP 99 CYCLES 1 HOLD

1. Sample(s) will be completed in 9 hours.

1. Turn on the cooling pump for the speedvac. Should warm up 20 minutes.
2. Remove 8 G-50 plates from refrigerator under Titer Tech Shakers.
3. Retrieve 8 used and 8 new Micro Amp 96 well Thermal Cycle Plates.
4. Label the 8 new plates with the correct sample id. Quadrant 1 PT is the first sample, quad 2 PT is the second, etc. Repeat with the JenRev plates. BE SURE TO NOTE PT OR JEN REV.
5. Set 4 G-50 plates onto 4 used Micro Amp 96 well Thermal Cycle Plates. It is critical plates are aligned correctly, otherwise water or sample will not be collected but lost in the centrifuge.
6. Spin 4 G-50 plates to remove water, PROGRAM 22.
7. Remove from centrifuge, decant water into sink (give it a good shake to get all the water out.)
8. Re-hydrate the plates again by adding 100ul autoclaved ddwater. Hydra Protocol: REHYDRATION G50 PLATES.
9. Spin again to remove water, PROGRAM 22.
10. While spinning re-hydrated plates, remove PT cycle sequence plate from the 4C refrigerator and add 10ul of water, Hydra Protocol: WATER ADDITION – 10UL TO RXNS IN 384 CS PLATE.
11. Remove G-50 plates from centrifuge and set on NEW 96 well Micro Amp Thermal Cycle Plates. Be sure they are labeled correctly.
12. Set the water reservoir to the side, move the PT CS plate to the source nest and set a G50 plate (from step 4) in the destination nest. It is critical that the PT CS plate and the G50 plate be placed in the correct orientation so that samples can be identified.
13. Transfer the reaction(s) from the PT cycle sequence plate to G-50 plate; Hydra Protocol: TRANSFER RXNS FROM 384 CYCLE SEQUENCE PLATE TO G-50.
14. Set loaded G-50 plate onto the labeled, corresponding NEW 96 well Micro Amp Thermal Cycle Plate and set in the centrifuge.
15. Repeat with remaining 3 PT samples. (Hydra will prompt you for the next plate.)
16. Spin, program 22, remove, set in black speedvac sample tray, and store in 4C until all samples are complete and ready for the Speedvac.
17. Repeat beginning with step 5 and JenRev Samples.
18. When PT and JenRev samples are ready, set in speedvac (uncovered; if only drying 4 plates, place them on the bottom rack to speed drying) and dry Speedvac 2-3 hours until dry. (Record time sample is placed into Speedvac and time removed on thermal cycling worksheets)

• Steps to initiate vacuum:
• Cooling pump on (warm up for 20 minutes.
• Place samples in Speedvac.
• Close cover on speedvac and cover top of Speedvac with foil.
• Turn the Concentrator ON.
• Turn vacuum pump power (under bench) ON.
• Initiate vacuum (turn bleeder valve).
• Check vacuum system for leaks.
• Check oil level on pump while pump is turned on, oil level should be between the 2 lines.
• Check the system, including the speedvac, pump and vacuum before walking away to be sure everything is working properly.
• Record time samples are placed in speedvac.
• Check in 2.5 hours and visually inspect for dryness.

1. When samples are dry, remove and cover with Corning aluminum foil seal.
2. Store at –20 until ready for 3700 analysis.