Labeling and Purification of Oligonucleotides with the
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1. gt COAN Mo 2002 CEQ 8000 Genetic Analysis System Labeling and Purification of Oligonucleotides with the Beckman Coulter Inc WellRED Dye labeled Phosphoramidites Beckman Coulter Inc 4300 North Harbor Boulevard Fullerton CA 92834 3100 Copyright 2002 Beckman Coulter Inc Copyright Licenses and Trademarks Beckman Coulter Inc 2002 All rights reserved No part of this publication may be reproduced transcribed transmitted or translated into any language in any form by any means without the written permission of Beckman Coulter Inc The software is copyrighted and may not be altered or given to a third party without the written authorization from Beckman Coulter Customer Support Please address all questions related to this product to DLPAhelp beckman com Include your name address and phone number in the body of your email IV Table of Contents Protocol for Labeling Oligonucleotides with Dye phosphoramidite on the ABI 392 or ABI 394 DNA RNA Synthesizer Required Reagents tea e Required Equipment and Materials esse Protocol ne ee Protocol for Labeling Oligonucleotides with Dye phosphoramidite on the Expedite DNA RNA Synthesizer Required Reagents cn Required Equipment and Materials esses PROROCO laicas Protocol for the purification of Dye labeled Oligonucleotides by Reverse Phase Cartridge Required Reagents oe beds oes Fee2. 0 1M TEAA pH 7 buffer Sample Loading 7 Pass the dissolved primer from step 6 through the cartridge at a rate of about 1 drop per second and collect the eluate 8 Pass the eluate from step 7 through the cartridge a second time in the same manner Cartridge Washing 9 Wash the cartridge 3 times with 5mL of 10 acetonitrile in 0 1M TEAA buffer pH 7 10 Wash the cartridge with 10mL of deionized water Sample Elution 11 Slowly pass drop by drop 1mL of 50 of acetonitrile deionized water v v through the cartridge into a microcentrifuge tube 12 Evaporate the purified sample solution until completely dry in a vacuum without heat e Use of heat during the drying steps will cause decomposition of the dye 13 Store the dry sample in the dark at 20 C until use Section IV Protocol for the purification of Dye labeled Oligonucleotides by Reverse Phase HPLC Please refer to the instrument s User s Manual for detailed operating instructions Required Reagents Description Part Quantity e en acetate TEAA buffer Glen Research 60 4110 57 a nssued 0 05M Triethylammonium acetate TEAA 0 5mL buffer pH 7 Buffer A 100 Acetonitrile EM Science AX0151 1 as needed Buffer B 50mM Ammonium Acetate buffer as needed pH 7 0 Buffer C 0 05M Triethylammonium acetate ashedddd buffer pH 10 TEAA HPLC water no DEPC as needed Required Equipme
3. a bust eee Required Equipment and Materials sess Protocol sen Cartridge Assembly auch Cartridge Pre load Preparation ocooonoccnnnccnonnnoonanoncnannnonnnono Sample Esadner c esci eisen CATING SS Was Fat e uoo s een Protocol for the purification of Dye labeled Oligonucleotides by Reverse Phase HPLC Required Redentor Required Equipment and Materials eese A O aoa ataw pada D Flow rate 1 5mL minute for all methods Ada 2 I 2 those 4 4 ta 7 EEEE 7 S 7 OE 7 dae fus 8 8 AEE LEEREN 9 TREE 9 Labeling and Purification of Oligonucleotides with the Beckman Coulter Inc WellRED Dye labeled Phosphoramidites The WellRED Dye labeled Phosphoramidites are cyanine based fluorescent dyes with high extinction coefficients that absorb in the near infrared spectral region These dyes were designed specifically for use with the CEQ series Genetic Analysis Systems and are excited to fluoresce using diode lasers much more stable and cost effective than traditional argon ion lasers The WellRED Dye labeled Phosphoramidites are easily coupled to the 5 end of oligonucleotides using commercial DNA synthesizers ABI 392 ABI 394 and Expedite The resulting labeled oligonucleotides may be used for direct hybridization or in PCR amplification processes Labeled DNA fragments may be detected quantitated and sized by the CEQ 8000 Gen
4. 1 500 29 5 0 0 70 5 0 0 6 28 00 1 500 100 0 0 0 0 0 0 0 4 29 00 1 500 0 0 100 0 0 0 0 0 6 30 00 1 500 2 0 0 0 98 0 0 0 8 34 00 0 000 2 0 0 0 98 0 0 0 11 Combine the labeled primer peak fractions into one 15mL conical tube then completely dry the eluate in a vacuum dryer without heat An example of an HPLC chromatogram with the labeled primer peak indicated is given below in figure 1 Use of heat during the drying steps will cause decomposition of the dye Resuspend the purified primer in 250uL of HPLC water no DEPO transfer to a microcentrifuge tube and then evaporate until completely dry in a vacuum dryer without heat 10 6 Repeat step 4 7 Store the dry sample in the dark at 20 C until use Figure 1 HPLC Elution Chromatogram 3 i E 7 s Dye Labeled Peak 4 4 kr I E j I v 8 4 y 8 n L Pj 2 j i 2 a E x iz z i3 S a amp A E amp 2 sz8 3 Ei z5 3 8 E pe 33 a Es 3 3 pon 5 2g SE 0 0000 Absorbance 11
5. PA to come to room temperature before use approximately 10 minutes This is to avoid moisture condensation that could damage the dye 3 Dissolve 100mg of the appropriate DLPA in 2mL anhydrous ACN DLPAs should be dissolved in super anhydrous ACN for 10 minutes at room temperature before installation on the DNA synthesizer 4 The Expedite DNA synthesizer instrument should be programmed to couple DLPA from position 5 6 or 7 as follows Time sec Function Mode Amount Argl Arg2 Description Deblocking 144 Index Fract Coll NA 1 0 Event out ON O Default WAIT 0 1 5 WAIT 141 Trityl Mon On Off NA 1 1 START data collection 16 Dbik PULSE 10 0 Dblk to column 16 Dbik PULSE 50 49 Deblock 38 Diverted Wsh A PULSE 40 0 Flush system with Wsh A 141 Trityl Mon On Off NA 0 1 STOP data collection 144 Index Fract Coll NA 2 0 Event out OFF Coupling 1 Wsh PULSE 5 0 Flush system with Wsh 2 Act PULSE 5 0 Flush system with Act 22 5 Act PULSE 10 0 Monomer Act to column 2 Act PULSE 1 0 Chase with Act 2 Act PULSE 4 600 10min Couple monomer 1 Wsh PULSE 2 31 Couple monomer 1 Wsh PULSE 30 0 Flush system with Wsh Oxidizing 15 OX PULSE 15 0 Ox to column 12 WshA PULSE 15 0 Flush system w
6. artridge Description Part Quantity Anhydrous Acetonitrile ACN HPLC grade EM Science AX0151 1 5mL nt A acetate TEAA buffer Glen Research 60 4110 57 5mL 0 1M TEAA buffer pH 7 1mL 1096 ACN in 0 1M TEAA pH 7 15mL 5096 ACN in HPLC water 1mL HPLC water no DEPC 10mL Required Equipment and Materials Description Partit Quantity Poly Pak cartridge Glen Research 60 1100 10 1 10 mL Disposable Syringe Becton Dickinson 1 Microcentrifuge tube as needed Pipettes 0 5 10uL 100 1000uL 1 each Savant Speed vac as needed Note Protocol Cartridge Assembly 1 Connect a syringe 10mL disposable syringe works well to the female luer of the cartridge and have the male luer terminate into waste 2 Make certain that all fittings are tight and secure Poly Pak cartridges are used for purification of oligonucleotides produced on a 50 nmole or 200 nmole scale pue Remove the whole syringe from the cartridge before removing the plunger The cartridge can be immobilized using a laboratory clamp Cartridge Pre load Preparation 3 Pass 5mL of ACN HPLC grade through the cartridge to waste 4 Pass 5mL of 2M TEAA Triethylammonium acetate buffer pH 7 through the cartridge to waste Sample Pre load preparation 5 Completely dry the dye labeled oligonucleotide after cleaving and deprotection with the appropriate reagent 6 Dissolve the primer residue in 1mL
7. etic Analysis System Absorbance and Emission spectral data Spectral Data Dye Absorbance maximum Emission maximum D2 PA 750 nm 770 nm D3 PA 685 nm 706 nm D4 PA 650 nm 670 nm Physical data Dye Phosphoramidite MW Dilution D2 PA 829 95 100 mg mL D3 PA 863 97 100 mg mL D4 PA 763 84 100 mg mL Absorbance Spectra of Dyes Emission Spectra of Dyes DA D3 D2 D4 D3 D2 0 90 f sso 600 650 700 750 500 680 700 720 740 760 780 800 820 840 Wavelength nm Wavelength nm Section Protocol for Labeling Oligonucleotides with Dye phosphoramidite on the ABI 392 or ABI 394 DNA RNA Synthesizer Please refer to the instrument s User s Manual for detailed operating instructions Required Reagents Description Part Quantity D2 PA 608147 100mg D3 PA 608146 100mg D4 PA 608145 100mg Pac dA CE phosphoramidite Glen Research 10 1601 xx as needed iPr Pac dG CE phosphoramidite Glen Research 10 1621 xx as needed Ac dC phosphoramidite Glen Research 10 1015 xx as needed Standard T phosphoramidite Glen Research 10 1030 xx as needed Anhydrous Acetonitrile ACN EM Science AX0151 1 1mL 0 05M Potassium Carbonate Methanol Glen Research 60 4600 30 1mL Ammonia Hydroxide J T Baker 9733 01 1mL HPLC water no DEPC 250uL Required Equipment and Materials Description Quantit
8. ith Wsh A Capping 12 Wsh A PULSE 30 0 End of cycle wash 1 Note that the final Capping process is omitted for the DLPA addition No Cap A or Cap B steps in the above program 2 Cleavage and deprotection of the oligonucleotide is performed by using NH4OH for 6 to 16 hours at room temperature in the case of D4 PA and D3 PA and by using 0 05M K2C0O3 MeOH for 8 16 hours at room temperature in the case of D2 PA The cleavage and deprotection is performed most efficiently by attaching the column to two syringes and mixing by pushing the solution back and forth between the syringes several times every 20 30 minutes Store the syringe column in the dark between mixing steps 3 After deprotection transfer the primer reagent mixture to a microcentrifuge tube and evaporate the reagent until completely dry in a vacuum dryer without heat Dissolve the primer in 300uL of HPLC Water no DEPC mix by vortexing and spin briefly in a microcentrifuge Evaporate the primer again until completely dry by placing in a vacuum dryer without heat ioe Use of heat during the drying steps will cause decomposition of the dye 4 Atthis point choose which method of purification Reverse Phase Cartridge or HPLC that will be used and proceed to the appropriate protocol for further instructions Section Ill Required Reagents Protocol for the purification of Dye labeled Oligonucleotides by Reverse Phase C
9. ligonucleotide is performed by using NH4OH for 4 hours at room temperature in the case of D4 PA and D3 PA and by using 0 05M K CO3 MeOH for 8 16 hours at room temperature in the case of D2 PA The cleavage and deprotection is performed most efficiently by attaching the column to two syringes and mixing by pushing the solution back and forth between the syringes several times every 20 30 minutes Store the syringe column in the dark between mixing steps After deprotection transfer the primer reagent mixture to a microcentrifuge tube and evaporate the reagent until completely dry in a vacuum dryer without heat Dissolve the primer in 300uL of HPLC Water no DEPC mix by vortexing and spin briefly in a microcentrifuge Evaporate the primer again until completely dry by placing in a vacuum dryer without heat Use of heat during the drying steps may cause decomposition of the dye At this point choose which method of purification Reverse Phase Cartridge or HPLC that will be used and proceed to the appropriate protocol for further instructions Section Il Protocol for Labeling Oligonucleotides with Dye phosphoramidite on the Expedite DNA RNA Synthesizer Please refer to the instrument s User s Manual for detailed operating instructions Required Reagents Description Partit Quantity D2 PA 608147 100mg D3 PA 608146 100mg D4 PA 608145 100mg Pac dA CE phosphoramidite Glen Re
10. nt and Materials Description Partit Quantity HPLC 1 Hamilton 79422 C18 HPLC column PRP 1 7um 250x4 1mm 1 each Microcentrifuge tube as needed 15mL conical tube as needed Pipettes 0 5 10uL 100 1000uL 1 each Savant Speed vac model 210A as needed Protocol 1 Resuspend the dried primer in 100uL of 0 05M TEAA pH 7 and pipette the solution into an appropriate HPLC cartridge or tube 2 Enterthe appropriate sample parameters and dye separation method from the chart below into the HPLC software 3 Purify the labeled oligonucleotide using the Hamilton PRP 1 Analytical HPLC column and separation buffers A B and C ONOaRWDND ONOAaARWBNDN o NOOA ON Flow rate 1 5mL minute for all methods D2 PA D3 PA D4 PA 4 5 Time Flow A B C D Curve 0 01 1 500 2 0 0 0 98 0 0 0 6 0 50 1 500 2 0 0 0 98 0 0 0 6 6 00 1 500 27 5 0 0 72 5 0 0 6 27 20 1 500 34 5 0 0 65 5 0 0 6 28 00 1 500 100 0 0 0 0 0 0 0 4 29 00 1 500 0 0 100 0 0 0 0 0 6 30 00 1 500 2 0 0 0 98 0 0 0 8 34 00 0 000 2 0 0 0 98 0 0 0 11 Time Flow A B C D Curve 0 01 1 500 2 0 0 0 98 0 0 0 6 0 50 1 500 2 0 0 0 98 0 0 0 6 6 00 1 500 28 0 0 0 72 0 0 0 6 27 20 1 500 36 0 0 0 64 0 0 0 6 28 00 1 500 100 0 0 0 0 0 0 0 4 29 00 1 500 0 0 100 0 0 0 0 0 6 30 00 1 500 2 0 0 0 98 0 0 0 8 34 00 0 000 2 0 0 0 98 0 0 0 11 Time Flow A B C D Curve 0 01 1 500 2 0 0 0 98 0 0 0 6 0 50 1 500 2 0 0 0 98 0 0 0 6 6 00 1 500 21 5 0 0 78 5 0 0 6 27 20
11. search 10 1601 xx as needed iPr Pac dG CE phosphoramidite Glen Research 10 1621 xx as needed Ac dC phosphoramidite Glen Research 10 1015 xx as needed Standard T phosphoramidite Glen Research 10 1030 xx as needed Anhydrous Acetonitrile ACN EM Science AX0151 1 1mL 0 05M Potassium Carbonate Methanol Glen Research 60 4600 30 1mL Ammonia Hydroxide J T Baker 9733 01 1mL HPLC water no DEPC 250uL Required Equipment and Materials Description Quantity Microcentrifuge tube as needed Pipettes Eppendorf 0 5 10uL 100 1000uL 1 each Expedite DNA synthesizer as needed Savant Speed vac as needed frost free freezer Protocol BCI Dye labeled Phosphoramidites D2 PA D3 PA and D4 PA phosphoramidites should be stored in the dark at 20 C in a non 1 Determine the scale of the synthesis and synthesize the desired oligonucleotide sequence using pac Ultramild amidites through the DMT On of the final base It is important to use the pac Ultramild amidites so that mild cleave and deprotection conditions can be used To achieve the highest labeling efficiency it is recommended that the oligonucleotides to be labeled be batched for labeling To do batch processing synthesize all of the oligonucleotides through the final base with the DMT On then reconstitute the appropriate DLPA and finish the labeling process 2 Allow the vial containing the dye labeled Phosphoramidite DL
12. y Microcentrifuge tube as needed Pipettes Eppendorf 0 5 10uL 100 1000uL 1 each ABI 392 or 394 DNA synthesizer as needed Savant Speed vac as needed a non frost free freezer Protocol BCI Dye labeled Phosphoramidites D2 PA D3 PA and D4 PA phosphoramidites should be stored in the dark at 20 C or below in 1 Determine scale of synthesis and synthesize the desired oligonucleotide sequence using pac Ultramild amidites through the DMT On of the final base It is important to use the pac Ultramild amidites so that mild cleave and deprotection conditions can be used To achieve the highest labeling efficiency it is recommended that the oligonucleotides to be labeled be batched for labeling To do batch processing synthesize all of the oligonucleotides through the final base with the DMT On then reconstitute the appropriate DLPA and finish the labeling process 2 Allow the vial containing the dye labeled Phosphoramidite DLPA to come to room temperature before use approximately 10 minutes This is to avoid moisture condensation that could damage the dye 3 Dissolve 100mg of the appropriate DLPA in 1mL anhydrous ACN The DLPAs should be dissolved in super anhydrous ACN for 10 minutes at room temperature before installation on the DNA synthesizer The DNA synthesizer instrument ABI 392 or 394 should be programmed for a 10 minute coupling period Cleavage and deprotection of the o
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