Lab Protocols: Quench Flow Burst Experiment (RT Example)


Operational procedures for the Quench-Flow

1. Turn on the thermometer and the water bath for 35°C to 100°C. Connect the 37°C water hose to the quench-flow chamber so the water can fill in.

2. Start mixing the enzyme/DNA and the dATP/Mg2+ to let them sit on ice for 20 min.

3. 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".

4. "Use constant quench volume (1/0)?" type in "1", "return."

5. "Enter second quench delay (sec)", type in "0", "return".

6. When the main menu pulls up, type "7" for seeking home position of the syringe driver.

7. Loading buffer: attach the 10ml buffer syringes - 1Χ RT buffer without Mg2+ to A and B, 500mM EDTA to C. Keep pushing in and out the plungers to get rid of the bubbles inside the quench-syringes. Change the syringe knobs to "fire" position.

8. 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.

9. Type "esc" to return to the main menu.

10. Fill the enzyme/DNA and dATP/Mg2+ mixes respectively into 1ml syringes. Plug the syringes into the designated load valves.

11. Wash the loops:

  • Change the knobs to "wash" position.
  • Turn on the vacuum.
  • Connect the vacuum tube to the sample exit loop.
  • Dip the 2 flush loops into ddH2O for 10s.
  • Dip into MeOH for 5s.
  • Let the loops dry for 15s.

12. On the main menu, type "1" for quench-flow run. "Enter the reaction time (sec)" type any value larger than 0.005s. "Return." Then the corresponding loop number will be displayed.

13. Switch the port to the required loop. Repeat the "wash" procedure.

14. Repeat the following quenching steps for each time point sample:

  • Enter the new reaction time. Push in the plungers of 1X RT without Mg2+ buffer syringes.
  • If a different loop is required, switch the loop, and "wash" again.
  • "Load" the enzyme / DNA mixture up the loop to just outside of the port.
  • "Load" the dATP/Mg2+ mix to just outside of the port.
  • "Fire": insert the exit loop into the prepared microfuge tube through the hole on the lid. Hit the "start" button on the controller or "G" on the keyboard. Put the collected sample into a radioactive tube rack.
  • "Wash", see step 11.

14. Exception: Do NOT load the dATP/Mg2+ when quenching at 0s time point

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15. When done with all the samples, enter "0" for reaction time to return to the main menu. Then enter "7" for home position of the syringe driver.

16. To discard the reaction mixes. Keep the vacuum on the exit loop. Switch to "load E/DNA*" and let the vacuum suck out the enzyme/DNA* mix. Unplug the 1ml syringe and immediately toss into the radioactive waste. Use a new 10ml syringe to drop ~10ml 0.2N NaOH into the insert rubble hole without plugging into it. Then drop ~10ml of 0.3N H3PO4 and then 20ml ddH2O and finally 1ml of MeOH.

17. Switch to "load dATP/Mg2+" knob position. Discard the 1ml syringe. Load in ~10ml ddH2O and then 1ml MeOH.

18. Press all 3 syringe-plungers to retrieve the 1Χ RT without Mg and 500mM EDTA buffer. Recap and store away the buffers.

19. Load 2× 5ml ddH20 into all 3 quench-syringes.

20. Change knob positions and turn on the vacuum to expel the ddH2O. Lastly flush all the loops by dipping the flush loops into ddH2O for 10s and MeOH for 5s. Let dry.

21. Turn off the controller and unplug the keyboard.

22. Turn off the thermometer and water bath. Disconnect the water hose to drain the quench-chamber.

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