SYNTHESIS PROTOCOL GUIDELINES when using ACTIVA UL1-EZFrit with Dr. Oligo Synthesizer

Transcription

1 SYNTHESIS PROTOCOL GUIDELINES when using ACTIVA UL1-EZFrit with Dr. Oligo Synthesizer A. Materials Needed.. p2 B. Important Reminders..p3 C. Strategies and Planning.. p4 D. Downloading Protocols at ctgen.com..p5 E. Summary of Protocol Parameters and Rules for Activa Method...p6 F. Summary of Protocol Parameters and Rules for using ACTIVA Frits with the Conventional Method....p7 G. Detailed Synthesis Parameters.p8 1. For ACTIVA UL1-EZ25 frit 2. For ACTIVA UL1-EZ100 frit 3. For ACTIVA UL1-EZ200 frit H. Degenerate Oligos or Wobble...p11 When finished, proceed to Step 6 : Cleavage and Deprotection of the 7 Steps to synthesize Oligos using ACTIVA frits 1

2 A. Materials Needed Materials Synthesis plate EZ columns Inserter/Extractor Molecular Sieving Pouches Reagents (click here for table) Materials Needed Additional Info/Comments Can be ordered from CTGen click here 10 columns are delivered free for 100 EZ-frits. Additional columns can be purchased click here Can be ordered from CTGen click here As moisture greatly diminishes synthetic yield, it is recommended to use molecular sieving pouches in order to keep water content in acetonitrile to less than 20 ppm. Pyridine-free reagents for ACTIVA Method may be ordered from reagent vendors (AIC, CTGen) or made in house by following the formulations table. All others reagents can be purchased from many reagent vendors such as AIC, Glen Research 2

3 B. Important Reminders Prior to Synthesis Please follow carefully those reminders which are crucial to your oligo synthesis success. Regarding ACTIVA Method Regarding Reagents for Activa Method click here for table Regarding Reagents for Conventional Method click here for table Regarding PUSH/DRAIN Pressure Settings Important Reminders 1. Note that the ACTIVA Method requires more care to set up than the Conventional Method. However, once routine is established, the ACTIVA Method provides significant reagent savings. 2. What is the Activa Method, Click here 1. Be sure to use pyridine-free reagents with ACTIVA Method. Formulations are available at (click here for table) 2. Keep a mm phosphoramidite concentration range. Do not lower or increase amidite concentration. 3. Do not use Activator. If use of Activator is desired, please use ACTIVA frits with the Conventional Method (click here) 4. For amidites, an additional 20% in recommended volume is tolerated but not advised. 5. Use only a 25 mm I 2 formula; higher iodine concentration will yield lower quality synthesis. 6. Do not use dichloroacetic acid (DCA) for the deblocking step; DCA is not compatible with the Activa Method. However, If the use of DCA is desired, use ACTIVA frits with the Conventional Method (click here) 1. Activator must be added (250 mm ETT or DCI). 2. Any reagents can be used, conventional and/or pyridine free reagents. 3. Use at least 50 mm phosphoramidite concentration solutions. 4. TCA or DCA can be used for the deblocking step. 1. Adjust the Push and HiDrain pressures to 1.5/2 and 5-6 PSI respectively depending on the kind of frit used. a. For EZ25 frit: 1.5 PSI for PUSH/ 5-6 PSI for DRAIN b. For EZ100 or EZ200 frit: 2 PSI for PUSH/ 5-6 PSI for DRAIN 3

4 C. Strategies and Planning for Reliable Oligo Synthesis Consistency and planning are the keys to reliable nucleic acid synthesis. Repetitive tasks organized into a Standard Operating Protocol streamline efficiency and produce better quality oligonucleotides: For Reliable Oligo Synthesis 1. Be sure Dr. Oligo is working properly by following our Daily Check-up list here Tips to Ensure Proper Dr. Oligo Performance 2. Start the synthesis. Confirm that the flow through the columns is not obstructed and watch the first few trityl releases. 3. Oligonucleotides of similar size should be combined in a same run. The difference in bases between the shortest and the longest should not exceed 10 bases. 4. Be sure there are enough of the required reagents available for the entire synthesis. Tips to Ensure Quality Reagents 5. Phosphoramidites that have been dissolved for more than 2 weeks should be discarded. 6. Exclude water from the system. Remove any stale reagents or moisture from the lines by priming them if the machine has been at rest for more than 30 minutes. This will maximize the first coupling step's efficiency. 4

5 D. Downloading Protocols at ctgen.com Download Protocols for ACTIVA EZfrit here Important Reminders About the Downloaded Synthesis Folders 1. Do not modify CTGen synthesis protocols and make sure to use the correct Dr. Oligo PUSH & DRAIN Pressures. 2. Strictly follow CTGen recommended reagents volumes and reagents concentrations. 1. There are three main synthesis folders: - EZ25 Activa Protocols - EZ100 Activa Protocols - EZ200 Activa Protocols 2. Each main folder contain two folders, PROTOCOL folder and MACRO folder: - The PROTOCOL folder contain protocols for Activa method AND protocol for Activa as Standard (i.e. conventional synthesis method) - The MACRO folder contains all needed macro to run the protocols. 3. The protocols were designed to fulfill the following goals: - Lower reagents consumption combined with - Average of 99.3 to 99.7 coupling yield efficiency. 4. To maintain the performance of the protocols, use them without any modifications. If you need to modify them, please read the Tips to build your own protocols section. 5

6 E. Summary of rules for Activa method Frit Priming Step Tips for Building ACTIVA Protocols Step/Rule 1 st base: 2X Ox and 2X Capping Steps Reagent Volumes Amidite Excess Push and Drain Pressures Description A minimum of two Deblock treatments is needed to remove the TMS protecting group from the UL1 linker and to fully protonate the support-bound Activator groups. Oxidation then Capping is carried out 2X to fully oxidize the 1 st base (see Q&A booklet) and to fully cap unreacted UL1 linkers. (learn why) Volumes described in the reagents volume tables are a minimum. Do not use less but do not use more than a 20% excess. Do not reduce or increase excess. Follow the Amidite Excess Table below. Follow the Pressures Table below for PUSH DRAIN. Amidite Excess & Volume used (for amidite solution with concentration at 17.5mM) - Pressure Table Kind of Frit EZ25 EZ100 EZ200 Amidite Excess / 8-10 X X 6-7 X volume used 30 l 50 l 70 l DRAIN (psi) 1.5/ 6 2/ 6 2/ 6 6

7 F. Summary of Rules for using ACTIVA Frits with a Conventional Synthesis Method Important Reminders: an external Activator (ETT, DCI ) is added during the coupling steps. Conventional reagents or pyridine free reagents can be used. Important Rules for Building Conventional Method Protocols Kind of Frit EZ25 EZ100 EZ200 Activator Concentration Amidite Concentration Activator volume ( l) Amidite volume ( l) 250 mm 50 mm Activa as Standard method protocols for conventional synthesis can be downloaded at ww.ctgen.com. 7

8 G. DETAILED SYNTHESIS PARAMETERS Stepwise details of synthesis parameters (reagent, volume added, Push time/reaction time, Drain time) are shown in Synthesis Parameter Tables. Note that parameters are specific for each kind of frit. The values showed here are good for a standard oligo quality; for specific oligo quality (gene synthesis, long oligo), some changes need to be made. 1. FOR ACTIVA UL1-EZ25 frit Important set up when using EZ25 frits: - PUSH pressure: 1.5 PSI / DRAIN pressure: 5-6PSI -! Set at 50 nmol scale at SYNTHESIS RUN PARAMETER (Dr. Oligo). ACTIVA UL1-EZ25 Pre- STEP REAGENT Vol added ( L) PUSH / wait #1 wait #2 wait #3 HIGH DRAIN Pre-Deblock 2X 1 st BASE CYCLE 3%TCA in Oxidation X2 Amidite I 2 (25 mm) Capping X2 CAP A/ CAP B 2 nd BASE & BEYOND CYCLE Deblock 3%TCA in Oxidation Capping Amidite I 2 (25 mm) CAP A/ CAP B 8

9 NOTE the following distinctions between methods: (1) Activa method to synthesize standard quality oligos up to 80-mer with no external addition of activator. (2) Activa method for gene synthesis to synthesize oligos used for gene synthesis, with extended reaction time for OX, CAP step. (3) Activa as Standard method for conventional synthesis with external addition of activator. 2. For ACTIVA UL1-EZ100 frit! Important set up when using EZ100 frit: - PUSH pressure: 2.0 PSI / DRAIN pressure: 5-6PSI - Set at 100 nmol scale at SYNTHESIS RUN PARAMETER (Dr Oligo). ACTIVA UL1-EZ100 STEP REAGENT Vol added ( L) PUSH / wait #1 (sec) wait #2 (sec) wait #3 (sec) HIGH DRAIN (sec) Pre /10 1/10 N/A 10 Pre-Deblock 2X 1 st BASE CYCLE 3%TCA in 300 1/30 2/30 2/ /3 1/3 1.5/3 8 Amidite /25 0.7/30 0.7/15 3 Oxidation X2 I 2 (25 mm) /5 0.7/10 0.7/5 3 Capping X2 CAP A/ CAP B 30/30 0.5/5 0.7/10 0.7/ /5 0.5/5 3/0 4 2 nd BASE & BEYOND CYCLE Deblock 3%TCA in /5 1/10 0.5/ /2.5 1/2.5 3/0 7 Amidite /25 0.7/30 0.7/15 3 Oxidation I 2 (25 mm) /5 0.7/10 0.7/5 3 Capping CAP A/ CAP B 30/30 0.5/5 0.7/10 0.7/ /5 0.5/5 3/0 4 9

10 NOTE: The following protocols are available for Activa UL1-EZ100 frits: (1) Activa method to synthesize standard oligos quality up to 70-mer. (2) Activa method for long oligo to synthesize oligos longer than 70-mer. (3) Activa as Standard method for conventional synthesis with external addition of activator 3. For ACTIVA UL1-EZ200 frit! Important set up when using EZ200 frit: - PUSH pressure: 2.0 PSI / DRAIN pressure: 5-6PSI - Set at 200 nmol scale at SYNTHESIS RUN PARAMETER (Dr Oligo). ACTIVA UL1-EZ200 Pre- STEP REAGENT Vol added ( L) PUSH / wait #1 wait #2 wait #3 HIGH DRAIN Pre-Deblock 2X 1 st BASE CYCLE 3%TCA in Oxidation X2 Amidite I 2 (25 mm) Capping X2 CAP A/ CAP B 2 nd BASE & BEYOND CYCLE Deblock 3%TCA in Oxidation Capping Amidite I 2 (25 mm) CAP A/ CAP B NOTE: The following protocols are available for Activa UL1-EZ200 frit: (1) Activa method to synthesize oligos up to 70-mer. 10

11 (2) Activa as Standard method for conventional synthesis with external addition of activator and pyridine-free or pyridine based reagents H. Synthesizing Degenerate Oligonucleotides or Wobble Dr. Oligo can be programmed for in-line degenerate mixing of bases instead of manually mixing the amidites together and using this mixture for the degenerate position. An EZfrit backpressure prevents entry of the in-line amidite mixture prior applying the pushing pressure. This allows for a thorough mixing of amidites ensuring that the degenerate position will not be skewed toward the phosphoramidite that is first delivered to the support. Thus, ACTIVA frits allow in-line mixing to generate all base substitutions at a given position with a uniform distribution of these substitutions. For detailed protocols, please inquire. 11