Difference between revisions of "DeRosa:Protocols/DNA synthesis"
(New page: ==Overview== To synthesize DNA on the MerMade 6 in 311 Steacie ==Materials== * amidites * CPG columns * anhydrous acetonitrile * CapA, Cap B * Activator * Oxidizer * Deblock * Argon * N...)
Latest revision as of 21:40, 1 October 2012
To synthesize DNA on the MerMade 6 in 311 Steacie
- CPG columns
- anhydrous acetonitrile
- CapA, Cap B
- 5 or 10 mL syringes
- Small vial if using modifiers
- 2 mL eppendorf tubes
- General DNA Synthesizer Notes
The synthesizer and Ar(g) should be left on at all times. (Without leaks, a full tank should last 1-2 months).
Ar(g) pressures should each read 5 +/- 0.5 psi. The synthesizer should be calibrated at 5psi approximately monthly. The SLPM should read 1.0.
Column vacuum should read 5mm Hg. If this needs to be changed for whatever reason (ie. modified columns), a vacuum calibration must be done.
DNA Synthesis Protocol
1) Write a “sequence file” in notepad with the following format: Sequence Name, Sequence (5’ to 3’). Save as a .txt file. Remember that the first base will be added manually during vacuum checking/calibration. So if the desired sequence was 3’-AGGTTGGTGTGGTTGGR-5’, choose an A column, choose standard mode, and G will be added during calibration. The sequence file will then be: Note if you are using a modifier (such as R in this example) it is recommended that you add this manually (and therefore it will not be in your sequence file). Also, if you are making sequences longer than 60nt, split the sequence file into 2 pieces.
2) Open the MerMade software: Click on ‘Set up run’. Select columns to be used and load corresponding sequence files. ‘Display CPG map’ will remind where to load columns. Choose ‘DMT on’ (leaves DMT protecting group at 5’-end), or ‘DMT off’ (performs a final deblock and wash) depending on application. If you are making the first part of a long sequence OR are adding a modifier manually be sure you leave DMT on. Select the script file which corresponds to the synthesis scale (usually 1umol) Using the ‘show total’ estimates, ensure that enough of each reagent is loaded on the synthesizer. Select amidite sizes that lead to the fewest bottles possible. Ex., if 0.75g is needed, use 1.0g, do not mix 0.5g+0.25g
3) Remove the necessary amidite bottles and columns from the -20C freezer, and place in the dessicator to come to room temperature. Can take up to 30 minutes.
Fill a syringe with argon to the required volume to make a 0.1M amidite solution (indicated in front cover of log book). To do this, remove the plunger from the syringe, let argon flow into the syringe. Replace the plunger and push the plunger to the required volume level. Inject this into the anhydrous acetonitrile diluent bottle, then remove the quantity of acetonitrile with the back pressure. Open the amidite bottle and as quickly as possible inject the acetonitrile and close the bottle. Swirl (and vortex) bottle until dissolved and load onto synthesizer ASAP. Repeat for all required amidites. If available, place the small trap pack into the amidite bottles as well.
From the injection screen of the software, ‘prime’ the amidites, activator, and other reagents. This means: press each inject button until no bubbles are visible in the corresponding lines leading to the chamber.
4) Return to the main screen of the software. Go to ‘service’, ‘motion options’, ‘load column’. Open the chamber and firmly, but not abusively, place the columns firmly in the correct positions.
5) Vacuum calibration (more often than not, this should be “just checking” if the vacuum strength is left alone, but for best results, must be done for each synthesis) Turn on the vacuum pump and return to the main screen. Go to ‘service’, ‘editors’, ‘edit script file’, choose the script file corresponding to the scale and modifiers used, click the amidite button that is first in your notepad sequence file (second in actual sequence, in the above example: G). Click ‘edit settings’, ‘edit current’, ‘calibrate valves’, and switch to the vacuum tab. Click each reagent and press the ‘inject’ button in the order of the phosphoramidite synthesis cycle:
ACN Wash Deblock Deblock (just to be sure) ACN Wash ACN Wash Amidite (note: machine automatically includes the Activator injection) ACN Wash Cap A (note: machine automatically includes the Cap B injection) ACN Wash Oxidizer ACN Wash x3
What you are looking for on each injection: That the level of liquid in the column always remains above the level of the upper plug, and that this level is just above the plug on the last pulse for that reagent. If the column dries out at any point other than the “draining and equalizing column” step, decrease the ‘vacuum pulse length’ parameter by a few milliseconds. If the reagent barely moves through the column at all during the pulses, and/or remains well above the plug on the last pulse, increase this parameter.
After the cycle is done, click ‘done’, ‘next’, and ‘finish’. For the remaining amidites, click ‘edit settings’, then instead of edit current, choose ‘copy from amidite x’, where x is the just calibrated amidite (G in this example).
Save the script file but add the date of synthesis to the filename (so that we all have the same base script file to go back to).
6) Return to Step 2, but this time, follow the prompts until finished and the ‘run’ option becomes available on the main screen. Click ‘run’.
Clean-up From the main screen, go to ‘service’, ‘Valve options’ ‘Injection head test’ and clear out all the leftover amidites (A,C,T,G, N, R) in the lines. Place amidite bottles containing ~5mL of cleaning acetonitrile onto the used positions, and run through the lines until dry. Clean the injection heads with a Kimwipe and acetonitrile. Ensure you clean the metal part that holds the injection tubing as well. Turn of the vacuum pump, and empty the waste into the DNA waste bottle in the fume hood (4L). When full, empty this into the steel waste container. Bring the steel waste container to Stores for disposal once it contains 8L. Wipe any spilled liquid from the column block and interior of the machine. NOTE: The argon (5psi) and machine remain turned ON. Do not remove the activator.
Table of Changes Person Change/Reason Comments Date Mike/Maureen Protocol drafted Sept. 27, 2011
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.