User manual / Technical Information
Contents
1. or Dissolve the PCR fragment in an appropriate amount of water or buffer avoid EDTA and EDTA containing buffers to adjust to ca 50 150 ng uL solution 2 Preparation of a 10 mM Label Azide L Ns solution select your preferred Oligo Click Azide combination from www carlroth com 21 Take 1 mg of your selected azide L N3 out of the freezer and slowly warm up to room temperature 2 2 Centrifuge shortly to place all L N3 on the bottom of the vial 2 3 Pipette 100 000 MW n3 uL of the click solvent into the vial containing the Label Azide 2 4 Vortex the vial until the Label Azide is dissolved completely 2 5 Centrifuge shortly 3 Performing the click reaction 1 2 min preparation 1 h reaction Be aware that the catalyst is solid and will not be dissolved during the click reaction Step 1 Pipette 3 uL of the activator yellow vial into the green vial Step 2 Pipette the appropriate amount of the oligo or DNA solution into the green vial from Step 1 Step 3 Pipette the correct amount of Label Azide solution reported in the Reaction Table at page 6 into the green vial from Step 2 Step 4 Gently vortex the green vial from Step 3 for 10 sec Centrifuge shortly Step 5 Place the green vial from Step 4 in a thermomixer at 45 C for 1h under gentle shaking do not exceed 700 rpi or in a water bath at 45 C for 1 h You can run the reaction at room temperature RT as well In this case use a prol2. 35 uL green 9 Reactors NA RT 1x 9x C Required Material and Equipment not provided with this kit e Alkyne modified oligonucleotide or Alkyne modified PCR fragment e Label Azide 10 mM e Centrifuge optional refrigerated e Microcentrifuge tubes e Thermomixer optional ROTI kit for DNA labeling Carl Roth GmbH Co KG e Ethanol 95 e 3M Sodium acetate solution 3 M NaOAc or ammonium acetate 3 M NH OAc D Work Flow Vial colour Name nen Add to the green vial Add the proper amount of oligonucleotide or DNA into the green vial Add the proper amount of Label Azide see Reaction Table into the green vial Gently mix the green vial Shortly centrifuge the green vial Heat to 45 C under gently shaking for 1h Alternatively place the green vial in a thermo bath for 1 h at 45 C Transfer the liquid phase in a new empty vial Wash the green vial with 60 pL NaOAc 3 M Transfer the liquid phases from the green vial into the new empty vial Add chilled EtOH 95 Proceed with your preferred work up Carl Roth GmbH Co KG ROTI kit for DNA labeling E Click protocol for Oligonucleotide and PCR labeling 1 Preparation of the Oligonucleotide or PCR fragment solution not provided with the kit Dissolve the oligonucleotide in the appropriate amount of water to adjust to a 0 1 1 mM solution and centrifuge shortly Also different concentrations can be used see Reaction Table at page 6
3. USER MANUAL Oligo Click S Reload ROTI kit for DNA labeling ROTI kit for DNA labeling Carl Roth GmbH Co KG Oligo Click S Reload ROTI kit for DNA labeling For Click Chemistry labeling of up to 10 nmol oligonucleotide containing 1 to 2 alkynes 9 Reactions For research use only Information in this document is subject to change without notice Carl Roth GmbH Co KG assumes no responsibility for any errors that may appear in this document Carl Roth GmbH Co KG disclaims all warranties with respect to this document expressed or implied including but not limited to those of merchantability or fitness for a particular purpose In no event shall Carl Roth GmbH Co KG be liable whether in contract tort warranty or under any statue or on any other basis for special incidental indirect punitive multiple or consequential damages in connection with or arising from this document including but not limited to the use thereof Cautions Activator amp lt gt Warning H226 H319 H335 P210 P280 P303 P361 P353 P305 P351 P338 P312a MSDS the appropriate MSDS can be downloaded from our website www carlroth com Literature citation When describing a procedure for publication using this product please refer to it as the Carl Roth s ROTI kit for DNA labeling Oligo Click S Reload We recommend using the following general protocol for click chemistry labeling of alkyne modified oligonucleotides up to 10 n
4. calculated in 2 4 e k equivalents of azide normally 1 30 Vazide Label Azide 10 mM Nazide pmol 10 nmol uL Add Vazide UL to your click reaction H Azide Table Use these tables to read out the minimum amount of Label Azide needed in your labeling click reaction in order to reduce the Label Azide consumption when needed For example if you have 7 nmol of an oligonucleotide nmol Oligo 7 containing 1 alkyne in the sequence Nr of Alkynes 1 then use 1 4 uL of the Label Azide 10 mM solution Nr of Alkynes 1 2 nmol Oligo uL Azide uL Azide 1 0 2 0 4 2 0 4 0 8 3 0 6 1 2 4 0 8 1 6 5 1 0 2 0 6 1 2 24 7 1 4 2 8 8 1 6 3 2 9 1 8 3 6 10 2 0 4 0 Troubleshooting If the labeling is not complete then increase the reaction time and eventually the reaction temperature recommended for multi labeling reactions and or for azides with high steric demand The amount of Label Azide reported in the Reaction Table at page 6 are for this example 4 0 uL which cover the range between 7 and 10 nmol oligo containing from 1 to 2 alkynes in the sequence Carl Roth GmbH Co KG ROTI kit for DNA labeling e Your notes 10 ROTI kit for DNA labeling Carl Roth GmbH Co KG e Your notes 11 Carl Roth GmbH Co KG ROTI kit for DNA lab
5. ona g mol 600 g mol x bp e 600 g mol is the average mass of a basepair e bp number of basepairs in your DNA template 2 3 Calculate the total amount of DNA now in nmol present in your sample Nowa nmol cona ng pL x Vona HL MW g mol e Com ng L measured in 2 1 e MWon g mol calculated in 2 2 e Vpn HL volume of your sample measure it with a pipette 2 4 Calculate the total amount of terminal alkyne modifications Nojynes in nmol in your DNA This amount corresponds to the amount of Thymidines in your DNA if dTTP was replaced by C8 Alkyne dUTP during PCR Nalkynes nmol bp x AT content 100 x Nowa nmol e bp number of basepairs in your DNA template e AtT content percentage of A s and T s in your DNA e Nowa nmol calculated in 2 3 If dCTP was replaced by C8 Alkyne dCTP during PCR then calculate nalxynes in nmol in your DNA as follow Nalkynes nmol bp x GC content 100 x Mona nmol e bp number of basepairs in your DNA template ROTI kit for DNA labeling Carl Roth GmbH Co KG e GC content percentage of G s and C s in your DNA e nova nmol calculated in 2 3 2 5 Calculate the amount of Label Azide nazide in nmol for labeling the alkyne modified DNA Labeling rates depend on the amount of Label Azide applied Normally 1 30 equivalents of azide are used resulting in labeling rates of up to 20 and more Nazide nmol Nalkynes nmol x k Nalkynes nmol
6. Label Azide although most of the reagents have been removed during the precipitation step Applicable purification methods 1 Desalting 2 RP HPLC 3 Gel Electrophoresis Carl Roth GmbH Co KG ROTI kit for DNA labeling Reaction Table Use the following table to calculate the amount of reagents Activator and Azide you need in your oligonucleotide labeling click reactions you in a fast and very reliable way You will need different amounts of Label Azide Azide uL Red column depending on the amount of oligonucleotide Oligo nmol range column and the amount of alkynes present in your sequence Alkyne content range column Add the reagents as described in Point 3 of this protocol 1f 70 Alkyne HL Activator uL Azide Reactor Minimal Maximal nmol content Yellow Red Green Oligo reaction range range Conc volume in uL 0 2 For a 22mer this range corresponds to 0 0 4 OD or 0 13 ug 1 2 3 1 0 S 0 1 mM 150 3 6 For a 22mer this range corresponds to 0 7 1 3 OD or 20 40 ug 1 2 3 2 5 S 0 1 mM 300 7 10 For a 22mer this range corresponds to 1 5 2 2 OD or 46 66 ug 1 2 3 4 0 S 0 1 mM 300 For a detailed calculation see page 7 of this user manual Use the Azide Table on page 9 in order to minimize the amount of Label Azide required in your labeling reaction ROTI kit for DNA labeling Carl Roth GmbH Co KG Appe
7. eight MW g mol of your oligo in order to obtain the total concentration in nmol l Multiply this value by the total volume in ul to obtain the total amount of your oligo Nojigo in nmol Example oligonucleotide containing two 2 alkynes and the following specifications e Coligo 250 ng uL e MWoligo 6500 g mol e Total volume Voligo 150 uL e Total amount Nojigo 250 6500 x 150 5 8 nmol Carl Roth GmbH Co KG ROTI kit for DNA labeling 1 3 Multiply Noligo by the total amount of incorporated alkynes in order to obtain Nalkynes in nmol e Oligo containing 2 alkynes Noligo 5 8 nmol e Nalkynes 5 8 x 2 11 6 nmol 1 4 The click reaction requires only two equivalents of azide Multiply Nalkynes X 2 to obtain Nazide In nmol e Nozide 11 6 x 2 23 2 nmol 1 5 Divide nazide by the azide concentration Cgzige 10 mM in order to obtain the amount of azide Vazide in UL to be used in the reaction Vazide Nazide Cazide 23 2 10 2 3 uL e Use 2 3 uL of Label Azide 10 mM in your click reaction 2 For PCR labeling Calculate the amount of Azide L N3 that you want to use for labeling your alkyne modified DNA The final labeling rate of the DNA can be tuned by the amount of azide used and has to be adjusted for every new DNA template 2 1 Measure the DNA concentration cpn4 ng L after PCR workup with a photometer 2 2 Calculate the molecular weight MW g mol of your DNA template MW png MW
8. eling Ordering information for detailed kit content see Table under B ROTI kits for DNA labeling seh Product Used Label Azide Reactor 7764 1 Oligo Click S Reload 9x2 5 mg 7765 1 Oligo Click M Reload 9x5 mg 7766 1 Oligo Click 5 488 6 FAM Azide 7 x2 5 mg 7767 1 Oligo Click M 488 6 FAM Azide 7x5 mg 7769 1 Oligo Click S 555 5 TAMRA Azide 7x2 5 mg 77701 Oligo Click M 555 5 TAMRA Azide 7x5 mg 77711 Oligo Click S Biotin Biotin Azide 7 x2 5 mg 77721 Oligo Click M Biotin Biotin Azide 7x5 mg To place your order please contact us e Phone 49 0 721 5606 0 e Fax 49 0 721 5606 149 e Email info carlroth com 12 ROTH Carl Roth GmbH Co KG Phone 49 0 721 5606 0 Schoemperlentra e 3 5 Fax 49 0 721 5606 149 76185 Karlsruhe Germany Email info carlroth com
9. mol with Label Azides provided by Carl Roth GmbH Co KG The Label Azides and the other auxiliary reagents can be ordered at Carl Roth GmbH Co KG separately Carl Roth GmbH Co KG ROTI kit for DNA labeling Protocol A General considerations e This protocol is optimized for the labeling of up to 10 nmol of a single or double alkyne modified oligonucleotide via copper l catalyzed azide alkyne cycloaddition CuAAc Click Chemistry e The Reactor S vial contains a stable heterogeneous catalyst which won t be dissolved during the reaction e The labeling reaction works more efficiently with concentrated solutions of alkynes oligo and azides Label Azide L N3 e The best way to carry out the click reaction is to mix the oligo and the Label Azide in a minimal amount of solvent e The click reaction is normally accelerated by elevated temperatures and can be finished in 30min when the reaction temperature is 45 C Low reaction temperatures e g 4 C can be applied as well in combination with longer reaction time e The reaction time depends on a concentration of azide and oligo in the solution b reaction temperature c stirring and or mixing of the solution d azide steric demand for double labeling reactions In the latter case use a prolonged 4 h reaction time B Materials and storage conditions for up to nine 9 independent labeling reactions provided with the Oligo Click S Reload Quantity Name
10. ndix F Calculation Sheet 1 Preparation of a 10 mM Label Azide L N3 Solution To calculate the amount of solvent V in uL to be added to 1 mg of Label Azide L N3 to prepare a 10 mM solution divide 100 000 by the molecular weight of the Label Azide MW yx E g e m Label Azide FAM N3 1 mg e MWin 458 4 g mol e V 100 000 458 4 218 2 uL e Cazide 10 mM 1 1 Take 1 mg Label Azide out of the freezer and slowly warm up to room temperature 1 2 Centrifuge shortly to place all the Label Azide on the bottom of the vial 1 3 Pipette V uL calculated in 1 of click solvent into the vial with the Label Azide 1 4 Vortex the vial until the Label Azide is dissolved completely 1 5 Centrifuge shortly This solution can be stored at 20 C in the dark for several months refer to the Label Azide Data Sheet The azide functionality is very stable and does not hydrolyze in water G Click reaction calculation sheet Use the Reaction Table on page 6 to read out the amount of Label Azide L N3 to be used in your experiment Use the Azide Table on page 9 if you need to minimize the amount of Label Azide used in your labeling reaction Below you can read how you can calculate those values yourself 1 For oligonucleotide labeling 1 1 Calculate the amount of oligonucleotide Nojigo in nmol Noligo mol m ng MW g mol e n nmol c mM x V uL 1 2 If you have a concentration c ng uL divide this value by the molecular w
11. onged reaction time 2 4 h This preparation is valid for Label Azides not included in this kit soluble in DMSO You can also use pure water or other solvents compatible with the Label Azide you selected see azides under www carlroth com This solvent contains a DMSO t BuOH mixture Download the MSDS from www carlroth com Art No 7815 3 See Minimal Oligo Conc and Maximal Reaction Volume in Reaction Table on page 6 See Reaction Table at page 6 or the calculation sheet on pages 7 9 ROTI kit for DNA labeling Carl Roth GmbH Co KG IMPORTANT Provide always some mixing over the reaction time The catalyst in the green vial will not be dissolved 4 Work up 15 20 min Step 7 4 1 Transfer only the liquid phase into a new empty vial 4 2 Wash the green vial containing the solid catalyst with 60 uL of 3 M NaOAc 4 3 Collect only the liquid phase from point 4 2 in the new empty vial containing your labeled oligonucleotide from step 4 1 Proceed with your preferred DNA precipitation or continue with point 5 5 Precipitation protocol Step 8 5 1 Add 1 mL cold ethanol 95 5 2 Centrifuge for at least 15 min at 4 C or cool for 1 h at 20 C and then centrifuge 5 3 Remove the supernatant and dry the residue on air 5 4 Re dissolve the pellets in the desired amount of water or buffer Your labeled oligonucleotide or DNA is ready for your experiment assay The final product may contain traces of free
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