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dU CPG Column

dU CPG Column

CPG column used to incorporate a deoxyuridine base into the 3' end of an oligonucleotide.

Key features

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  • Used to induce mutagenic effects.
  • The enzyme uracil-N-glycosylase (UNG) can specifically remove uracil to create abasic sites at the deoxyuridine positions. This property is used to generate site-specific strand breaks in the oligonucleotide.
  • CPG has long-chain alkylamino succinyl linker.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Product information

Incorporation of a deoxyuridine base within a DNA sequence can be used to induce mutagenic effects. The enzyme uracil-N-glycosylase (UNG) can specifically remove uracil to create abasic sites at the deoxyuridine positions. This property is used to generate site-specific strand breaks in the oligonucleotide. We provide both dU phosphoramidite and CPGs. 

Synthesizer
Column
Type/Description
Notes
MerMade 6,12
MerMade, syringe (all scales)
Pipette type column
A MerMade column is also known as a Supercolumn
MerMade 4, 48X, 96E, 192E, 192X
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn
ABI 384 / 394
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
Expedite 8909
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
ABI3900
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
K&A H4, H8, H8SE, H2, H32, H64
Luer
Barrel column with luer fitting at either end
Also known as ALL-FIT or Standard
K&A S4CL/S8CL
Luer
  Barrel column with luer fitting at either end 
 Also known as Standard. For this instrument, we recommend the Luer (Standard) column as the ALL-FIT columns have a wider barrel.
Dr Oligo 48
MerMade
Pipette type column
A MerMade column is also known as a Supercolumn
 Dr Oligo 192XLc, 768XLc just plates 
 MerMade, Syringe (up to 1.3 mL) 
Pipette type column
A MerMade column is also known as a Supercolumn
 OligoMaker X12, 48, 192, X192, X96 
MerMade, Syringe (up to 1.3 mL)
Pipette type column
A MerMade column is also known as a Supercolumn

Applicable Products

LK2013 dU-CE Phosphoramidite
LK2016 dI-CE Phosphoramidite
LK2017 5-Me-dC(Bz)-CE Phosphoramidite
LK2145 2-Amino-dA-CE Phosphoramidite
LK2164 2'-Deoxyxanthosine-CE Phosphoramidite
LK2287 dU SynBase™ CPG 1000/110
LK2293 dI SynBase™ CPG 1000/110
LK2323 5-Me-dC SynBase™ CPG 1000/110
LK2529 5-Me-dC(Ac)-CE Phosphoramidite

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK2013 C39H47N4O8P 730.80 290.17 3.42 6.84 13.68
LK2016 C40H47N6O7 754.83 314.19 3.31 6.62 13.25
LK2017 C47H54N5O8P 847.90 303.21 2.95 5.90 11.79
LK2145 C58H83N10O6P 1047.33 328.22 2.39 4.77 9.55
LK2164 C56H61N8O12P 1069.12 330.20 2.34 4.68 9.35
LK2287 - - 290.17 - - -
LK2293 - - 314.19 - - -
LK2323 - - 303.21 - - -
LK2529 C42H52N5O8P 785.88 303.21 3.18 6.36 12.72

Coupling

Couple the phosphoramidites LK2013, LK2016 and LK2017 using the standard method as recommended by synthesiser manufacturer with coupling times as per standard bases. 90s is recommended for 2529. A coupling time of 15min is recommended for LK2145. A coupling time of 3min is recommended for LK2164. The solid supports are used as per standard nucleoside supports.

Deprotection

For LK2013, LK2016, LK2017 and LK2529 standard oligonucleotide deprotection conditions can be applied when deprotecting an oligo containing these modifications, however LK2013, LK2016 and LK2529 are also compatible with AMA deprotection; typically 10min at 55oC.

LK2145 - Use AMA deprotection in conjunction with Ac-dC, rather than Bz-dC, or transamidation will occur. The supports are cleaved and deprotection carried out using the protocols required by the nucleobases.

LK2164 - Cleavage from support and primary deprotection is accomplished by treatment with ammonium hydroxide solution at room temperature for 24h.

After removing the solution1, the NPE groups are removed by treatment with 0.3M tetramethylguanidinium 2-nitrobenzaldoximate solution in water/dioxan (1:1) at 70°C for 48h. We find that this extended treatment is necessary to ensure complete removal of both of the NPE protecting groups.

It should be noted that this treatment generates a variety of low molecular weight by-products which are observed in the HPLC. Satisfactory results are obtained by de-salting the deprotection mixture into water using a NAP or G25 column before HPLC, from which the final oligonucleotide product can then be isolated.

Storage & Stability

Refrigerate dry compounds. Stability of phosphoramidites in solution is similar to standard dA, dC, dG and dT monomers.

Note

  1. This is best achieved by removing the deprotection solution by G25 then freeze drying to remove the water. Heat cannot be used or the NPE groups would cleave.

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