Protocol

Rapid quench operational procedures

Preliminary procedures

1. Prepare buffers.
2. Turn on the thermometer and the water bath for 25 °C
3. Start mixing the enzyme/DNA and the dNTP/Mg²⁺ to let them sit on ice for 20 min.
4. Turn on the controller. Plug in the adapter cord to the keyboard. Disregard the message "system error 5" if it appears. Type in "RUN," "return"
5. When the main menu pulls up, type "7" for seeking home position of the syringe driver.
6. Load Syringes.
   1. Turn syringe knobs to Load position.
   2. Add 1 Reaction Buffer without Mg²⁺ to left and right syringes.
   3. Load 500 mM EDTA (pH 8.0) to Quench syringe.
   4. Keep pushing in and out the plungers to get rid of the bubbles inside the syringes
   5. Change the syringe knobs to "fire" position.
7. Type "2" for adjusting position of the syringe bar. Press "start" button on the controller to lower (+, default) or raise (-) the bar. Keep lowering the syringes till the buffers start coming off the exit loop.
8. Type "ESC" to return to the main menu.
9. Fill the enzyme/DNA and dNTP/Mg2+ mixes respectively into 1 mL disposable syringes. Plug the syringes into the designated sample load valves.
10. Switch the port to the required loop. Repeat the "wash" procedure.

Collect time point

1. On the main menu, type "1" for quench-flow run.
2. "Enter the reaction time (sec)" type any value larger than 0.002s. "Return." Then the corresponding loop number will be displayed.
3. Determine which reaction loop is required
4. Move to new reaction loop.
5. Wash the loops.
   1. Turn on vacuum
   2. Quench knob to off position. Left knob to off. Right knob to flush.
   3. Place H₂O under right flush line.
   4. Attach vacuum to center line (sample exit loop)
   5. Let H₂O flow for 10 sec.
   6. Move H₂O from right line to left line
   7. Left knob to flush. Right knob to off.
   8. Let H2O flow for 10 sec.
   9. Place right line in MeOH
   10. Right knob to flush. Left knob to off.
   11. MeOH for 5 s
   12. Move MeOH from right ot left line.
   13. eft knob to flush. Right knob to off.
   14. MeOH for 5 s
   15. Right knob to flush
   16. Remove MeOH
   17. Let lops dry for 15 s.
6. Turn Sample Values to load.
7. Load DNA/enzyme into sample load loop
8. Load dNTP/Mg²⁺ into opposite sample load loop.
9. Turn Sample Valves to Fire.
10. Check that all valves are in Fire position.
11. Place microcentrifuge tube onto end of sample exit loop.
12. Press "G" to run reaction.
13. Remove microcentrifuge tube.
14. Go to step 2.
15. When done with all the samples, enter "0" for reaction time to return to the main menu.
16. Then enter "7" for home position of the syringe driver
17. Exception: Do NOT load the dATP/Mg²⁺ when quenching at 0s time point.

Wash up.

Wash syringes.

1. Turn Syringe valves to Load.
2. Press all 3 syringe-plungers to retrieve the 1X Reaction Buffer without Mg²⁺ and 500mM EDTA buffer. Recap and store away the buffers.
3. Wash syringes with H₂O three times.
4. Fill syringes for a fourth time and switch Syringe knobs to Fire.
5. Push H₂O through each of the reaction loops.

Sample loading loops

1. Remove dNTP/Mg²⁺ and enzyme/DNA syringes
2. Switch Enzyme/DNA knob to load.
3. Place waste tube under Sample /exit Loop.
4. Wash with
5. 10mL 0.2N NaOH
6. 10mL of 0.3N H₃PO₄
7. 20mL H2O
8. 1mL of MeOH.
9. Turn knob to Fire.
10. Turn dNTP/Mg²⁺ knob to load.
11. Wash with
12. 10 mL H₂O
13. 1 mL MeOH
14. Turn knob to Fire.

Wash Reaction Loops

1. Change knob positions and turn on the vacuum to expel the ddH₂O.
2. Lastly flush all the loops by dipping the flush loops into ddH₂O for 10s and MeOH for 5s. Let dry.
3. Turn off the controller and unplug the keyboard.
4. Turn off the thermometer and water bath.
5. Disconnect the water hose to drain the quench-chamber.