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TAMRA-Phos-CPG Column (5'-DMT-mdC(TEG-TAMRA)-Phos-CPG)

TAMRA-Phos-CPG Column (5'-DMT-mdC(TEG-TAMRA)-Phos-CPG)

A CPG column used for 3'-TAMRA (rhodamine) labelling of an oligonucleotide, with a TEG spacer.

Key features

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  • Used to add a broad range green-blue quencher to the 3' end of an oligonucleotide.
  • Will quench FAM, HEX, TET and JOE; perfect for dual-labelled probes and probe-based assays.
  • Also has fluorophore properties, emission maximal at 580 nm.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Product information

TAMRA (carboxytetramethylrhodamine) exists as 5- and 6-derivatives, or as a mixture of both isomers. It is the most commonly used rhodamine dye in oligonucleotide based applications. The fluorescent properties of TAMRA are sometimes used in non-radioactive sequencing, and in situ hybridisation studies, however TAMRA is more often as a quencher in Real-Time PCR applications based on FRET. The light-absorbing properties of TAMRA, and spectral overlap with several commonly used fluorophores - including FAM, HEX, TET and JOE, make it useful as a quencher for the design of dual-labelled probes. The usefulness of TAMRA is, however, limited because of its broad emission spectrum, which reduces its capabilities in multiplexing. Its intrinsic fluorescence contributes to the background signal, potentially reducing the sensitivity of assays based on TAMRA.

Despite these limitations, TAMRA has been used extensively in the design of probe-based assays, perhaps most notably in Taqman probes for Real-Time PCR. TAMRA is not sufficiently stable to strong bases; the molecule degrades in the presence of ammonium hydroxide, hence TAMRA-labelled oligos must be synthesised using mild deprotection monomers. Subsequent deprotection of the oligo is achieved with t-butylamine/methanol/ water (1:1:2) for 2.5h at 70˚C. Although there is still a small amount of TAMRA degradation, this is easily removed during purification. Alternatively, amino-modified oligos can be post-synthetically labelled using a suitable TAMRA NHS ester. We provide a number of solid support and phosphoramidite options for direct TAMRA labelling internally, or at the 3’ or 5’ end of oligonucleotides.

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

Properties:

  • Formula: C77H91N8O21PS
  • Molecular Weight: 1527.63
  • Appearance: pink powder
  • Absorption Maximum (Lambda Max): 555
  • Extinction Coefficient at Lambda max: 90000
  • Extinction Coefficient at 260 nm: 31980
  • Fluorescence Maximum: 576

Spectral properties measured in PCR buffer as 3'-labeled poly(T) oligo.

Product usage:

  • Cleavage conditions: Use t-butylamine/water (1:3) for 45 minutes at 25 °C. The use of ammonia or methylamine must be avoided, as it will cause degradation of the TAMRA moiety.
  • Deprotection conditions: When using fast deprotecting amidites (eg. C-Ac, G-DMF, G-PAC) use t-butylamine/Water (1:3) for 6 hours at 60° C. When using standard amidites (eg. C-Bz, G-iBu) use t-butylamine/Water (1:3) overnight (12-15 hours) at 60°C.
  • ABI 3900 users: Columns should be compatible with standard ABI protocols. However, when using 50 nmol Super Columns, the following changes are recommended: 1) Change the head pressure/purge pressure/chamber pressure to 3.5 psi.2) An additional oxidation step.3) OPTIONAL (but may help): extension of the coupling time to 20 sec.Image of cleaved and deprotected structure:
  • The mass this product adds after conjugation and work-up (the additional mass seen by mass spectrometry) is: 998.91

Storage and handling:

  • Shipping conditions: Cold
  • Storage conditions: -15 to -30 °C in sealed container

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