Subject: Cycle Modification on the ABI 3900 DNA Synthesizer using software v1.2

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1 Subject: on the ABI 3900 DNA Synthesizer using software v1.2 Overview: This bulletin describes how to modify the cycle files on the ABI 3900 DNA Synthesizer. In this User Bulletin: Topic Page Cycle File Layout 1 Creating a Purge Label 2 Changing Iterations 3 Changing Volume 3 Using the WAIT field 4 Modifying Coupling Wait Time 4 Additional Reagents 4 Adding Steps to the Cycle Loop 6 Adding Steps after End Loop 7 Simulation Mode 8 Caution: The ABI 3900 DNA Synthesizer is a very flexible instrument and often the user may wish to make modifications to cycle files in order to take advantage of the flexibility. The modifications discussed in this bulletin are for demonstration purposes only and are not optimized cycles provided by Applied Biosystems. This bulletin is intended as a guide to cycle modification, and the user will be responsible for the modifications that are made. Cycle File Layout: The cycles are in Microsoft Excel spreadsheet format that allows for easy modification. The cycle files are found in the 3900Cycles folder that is within the 3900 DNA Synthesizer folder. They are named according to the synthesis scale, phosphoramidite concentration and revision. The example shown below is file: 200nm, 0.05M, Rev E.xls. The files are "Read Only" so you will need to save it under another name in order to modify it. All times are entered as seconds and all volumes are entered as microliters. The cycle is divided into two parts: Upper Case and Lower Case. If an upper case letter is used in a sequence then the upper case method will be performed for that base. Likewise, if a lower case letter is used then the lower case portion of the cycle file will be performed. There are six different headings: Purges, # Iterations, Wait sec., Command Code, Param1 Chemical and Param2 Volume. The purges are common to the upper and lower case method. If a new purge is selected it will affect both the upper and lower case procedures. Aside from the two methods, there is a Coupling Wait field that may be edited and this coupling time pertains to the phosphoramidites in the regular A, C, G and T positions.

2 The modified amidite bottle positions, 5,6,7,8,9,0 follow the upper case procedure with the exception of the coupling step. The coupling step for each bottle position is separately programmed using the headings Volume, Reps, Wait and Cpl. Wait under the section titled, Coupling of Modified Amidites. Creating a Purge Label: A purge is defined as the time the waste valves are opened so essentially it is the time allowed for the columns to drain. The Purge settings may be edited or new purges created. A purge may be carried out in several repetitions. The purge settings are given a name, then the number of repetitions (Reps), followed by the seconds per Rep, and finally an Interim time may be added. The Interim is the length of time between Reps. Vary the Seconds time depending on whether you want to pull everything through the column or if you simply want to load reagent onto the column. For example, during a REACT purge it is good to have a short time since the coupling wait time occurs after the REACT purge. The purpose of the REACT purge is to load the activator and phosphoramidite onto the column. The purpose of the remaining purges in the default file is to drain the columns. Tip: If you increase the repetitions for REACT purge and enter an interim time then it may be advantageous to decrease the coupling time accordingly. For repeat REACT, it is

3 recommended to use a tenth of a second and repeat that two or three times to prevent liquid from draining completely through the columns. The new purge label can be used in the cycle procedure as shown below or use it as the new purge label for mixed mode amidites. Changing Iterations: The number of iterations for any step may be changed by editing the number in the # Iterations field for upper and lower case procedures or in the Reps field for modified amidites and purge settings. For example, to double couple in the upper case method, change the entry in cell C16 from 1 to 2. The # Iterations refers to the number of times that step (addition followed by purge) is performed. If the iteration is 2 then the instrument will perform the following operations: dispense, purge, dispense and purge. To change the number of times a modified amidite is coupled, edit the number in the Reps column that corresponds to that bottle position. The default coupling repetition is 2 for positions 5,6,7,8,9,0. Changing Volume: Editing the number under the Param2 Volume heading would alter the dispensing volume for each reagent. This is the volume to be dispensed to each column per iteration. These values may be increased or decreased. The volumes for the specialty bottle positions are listed in the orange box at the bottom of the file.

4 Using the WAIT Field: Some applications may require that a wait time be inserted before a purge. In the upper and lower case procedures the Wait sec. column may be used for this purpose. The specialty bottle positions also have a Wait field. An alternative to using a Wait time is to create a new purge label and use multiple repetitions with an Interim wait time. This ensures that the reagents are loading onto the column. Modifying Coupling Wait Time: To modify the coupling wait time, change the number in the Coupling Wait Time field. The default times are 40 seconds. The coupling wait time for the specialty bottle positions are found in the column, Cpl. Wait. Additional Reagents: The 3900 synthesizer allows the user to: 1. Take advantage of the specialty bottle positions for additional reagent delivery 2. Substitute existing reagent positions with non-standard reagents. Whenever a different solution is placed on the instrument, the valves that will be delivering the new reagent must be re-calibrated. Using Specialty Bottle Positions for Additional Reagent Delivery: Modify the cycle file by typing the bottle number in the field where you want to substitute that reagent. Place your reagent on the instrument, prime the lines and calibrate the valves. In the example below, the lower case procedure will add reagent from bottle 5 instead of OXIDIZER.

5 Substituting existing reagent positions with non-standard reagents: The specialty bottle positions may not provide enough volume for some applications. An existing reagent may be substituted for a new reagent by: 1. Changing the name in the cycle file at the appropriate step. Use upper case letters only. 2. Change the name in the Valve Configuration Map in the 3900 synthesizer software. This can be found in Instrument>Valve Configuration. The name must be spelled exactly as it is in the cycle file.

6 Reagent names must match the Param1 Chemical names in the Cycle file Adding Steps to the Cycle Loop: The best method for adding steps to the cycle file is to copy the 2 lines below, then insert them into the spreadsheet. Then modify the step as necessary. The important point to remember is that the rows in the upper case procedure must match the rows in the lower case procedure. If the application requires extra steps in one of the procedures only, then put extra washes into the other procedure.

7 An alternative is to fill the empty cell by breaking up the DISPENSING into two steps as shown below. Add new step Add OXIDIZER in 2 steps (2 x 30µL) Adding Steps After End Loop: Similar to the discussion above for adding steps within the loop, steps may be added after the End Loop.

8 Simulation Mode: When editing cycles it is often a good idea to run the instrument in simulation mode using the edited cycle. Not only will it confirm that the 3900 synthesizer software will accept the cycle file but the user may view the log file to ensure that additions, purges, etc occur in the intended order and that all steps are executed. To use simulation mode, in the 3900 software, go to Tools>Options and uncheck the motion and dispensing boxes. (Be sure to recheck those boxes before performing a real synthesis.)

9 THIS MATERIAL OR SPREADSHEET I S PROVIDED AS IS, WITHOUT ANY EXPRESS OR IMPLIED WARRANTIES WHATSOEVER, INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, WHICH ARE ALL HEREBY EXPRESSLY DISCLAIMED BY APPLIED BIOSYSTEMS. APPLIED BIOSYSTEMS SHALL NOT BE LIABLE FOR ANY CONSEQUENTIAL. INDIRECT, PUNITIVE OR SPECIAL DAMAGES IN CONNECTION WITH THE USE OF THIS MATERIAL OR SPREADSHEET EVEN IF APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY THEREOF. Notice to Purchaser: License Disclaimer Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation, either expressly, or by estoppel. Applied Biosystems is a registered trademark and AB (Design), Applera, and ABI are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. All other trademarks are the sole property of their respective owners.

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