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rG (iPr-Pac) CPG Column

rG (iPr-Pac) CPG Column

CPG column for incorporation of unmodified ribo-G at 3' end of an oligonucleotide.

Key features

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  • CPG has long-chain alkylamino succinyl linker
  • Phenoxyacetyl (Pac) protection for use in UltraMILD conditions
  • ALL-FIT luer or MerMade pipette columns available
  • Available in different synthesis scales
  • Available indivdually (minimum quantity of 10) or in packs of 10
Option 1: Select a Pore Size
Option 2: Select a Column Type
Option 3: Select a Scale
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Product information

Various RNA CPG column formats are available, and compatible with a range of instruments. 

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

LK2033 dmf-G-CE Phosphoramidite
LK2036 Bz-A-CE Phosphoramidite
LK2037 Pac-A-CE Phosphoramidite
LK2038 Ac-C-CE Phosphoramidite
LK2039 iPr-Pac-G-CE Phosphoramidite
LK2040 U-CE Phosphoramidite
LK2053 Ac-G-CE Phosphoramidite
LK2091 5-Me-U-CE Phosphoramidite (ribo-T)
LK2295 U-SynBase™ CPG 1000/110
LK2309 Ac-C-SynBase™ CPG 1000/110
LK2318 dmf-G-SynBase™ CPG 1000/110
LK2319 Pac-A-SynBase™ CPG 1000/110
LK2320 iPr-Pac-G-SynBase™ CPG 1000/110
LK2321 Bz-A-SynBase™ CPG 1000/110

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

LK2033 C49H67N8O8PSi 955.18 345.21 2.62 5.23 10.47
LK2036 C53H66N7O8PSi 988.21 329.21 2.53 5.06 10.12
LK2037 C54H68N7O9PSi 1018.23 329.21 2.46 4.91 9.82
LK2038 C47H64N5O9PSi 902.11 305.18 2.77 5.54 11.09
LK2039 C57H74N7O10PSi 1076.31 345.21 2.32 4.65 9.29
LK2040 C45H61N4O9PSi 861.06 306.17 2.90 5.81 11.61
LK2053 C48H64N7O9PSi 942.14 345.21 2.65 5.31 10.61
LK2091 C46H63N4O9PSi 875.09 320.19 2.86 5.71 11.43
LK2295 - - 306.17 - - -
LK2309 - - 305.18 - - -
LK2318 - - 345.21 - - -
LK2319 - - 329.21 - - -
LK2320 - - 345.21 - - -
LK2321 - - 329.21 - - -

Coupling

The steric hindrance introduced by the additional 2’-protecting group reduces the coupling efficiency of the phosphoramidite, and is limited to >97% using tetrazole activator even with extended reaction times. When using 0.25M ETT (LK3140/LK3142), to optimise efficiency a 12min coupling time is recommended. It has been shown that this coupling time can be reduced to 3min when using 0.3M BTT as the activator (LK0234/LK3160/LK3162).

Cleavage & Deprotection

Removal of the base protecting groups, cleavage of the linkage to the support and removal of the ß-cyanoethyl groups are all achieved routinely with aqueous 20% NH3/methylamine (1:1). The cleavage is fast (10-30min) at 65°C. (Heating can be continued up to 1h, however prolonged treatment will cause gradual loss of TBDMS groups resulting in degradation.) The deprotection strategies used are dependent on the base-protection utilised. Bz-A (LK2036), dmf-G (LK2033), Ac-C (LK2038) and Ac-G (LK2053) can also be deprotected using AMA (1:1 ammonium hydroxide solution in aqueous methylamine) at 65°C for 10min, however it should be noted that the use of ethanol in the deprotection solution aids solubility for full RNA sequences. When synthesising RNA chimera, e.g. RNA/DNA or RNA/2’-OMe RNA, the C amidite cannot be Bz protected otherwise deprotection with AMA will result in transamidation.

Pac-A (LK2037), iPr-Pac-G (LK2039) and Ac-C (LK2038) can be deprotected using UltraMILD conditions such as 0.05M potassium carbonate in methanol at room temperature for 4h.

Desilylation

The desilylation procedure is the same for DMT ON and DMT OFF oligos. Suspend the residue in dry N-methyl pyrrolidone/Et3N/Et3N.3HF (6:3:4 v/v/v) and deprotect silyl groups for 2.5h at 65°C in a sealed sterile tube. Alternatively, DMSO (or DMF)/Et3N.3HF (3:1 v/v) can be used.

Detritylation & Purification

Retaining the DMTr group after oligonucleotide synthesis is advantageous to the purification of RNA. This allows failures with no 5’-DMTr protection to be easily removed from the crude mixture. Traditionally this was carried out by RP-HPLC where the full-length DMTr-protected oligonucleotide was collected then detritylated using 10% acetic acid at pH 3.5 (30-45min) then quenched with ammonium bicarbonate followed by desalting.

Today it is possible to carry out the purification by RP-HPLC which includes the detritylation step and the full-length detritylated product is collected. Also, cartridge purification - e.g. TOPS, PolyPak or GlenPak - allow fast processing of oligonucleotides where DMTr-oligos are loaded onto the column and the full-length detritylated product eluted. This is particularly useful in preventing cross contamination since a new column is used for each oligo. DMT-ON purification, although useful, does not remove the N-1 sequences where the DMTr protection is retained. In this case cartridge purification can be useful to remove all other failures prior to purification by IE-HPLC. The latter is also the recommended method for purification of RNA where the DMTr group has been removed, or where the sequence is longer (≥40 bases). The most commonly used IE columns for purifying RNA are Dionex DNA-PAC (100 or 200) columns although there are alternatives from Phenomenex and other suppliers.

Typical buffers are:

Anion-exchange HPLC—A: 20mM aqueous Tris-HCl pH 7.6 + 1mM EDTA + 10mM sodium perchlorate; B: As A with 600mM sodium perchlorate. This system may require alteration for longer oligos.

RP-HPLC—A: 95% 0.1M triethylammonium acetate (TEAA) + 5% acetonitrile; B: 95% acetonitrile + 5% water (or 5% 0.1M TEAA). Gradient of 0-50% B during 20min.

Much longer oligos are best synthesised DMT-OFF and purified by PAGE.

Storage & Stability

All solid phosphoramidites and RNA supports are stored dry in a freezer at –10 to –30°C. The phosphoramidites are stable in anhydrous acetonitrile solution for 24h (LK2091 can be stored for 2-3 days).

Unlike DNA, RNA is highly unstable in basic media and must not be exposed to high pH. Traces of heavy metal ions present in various salts used also lead to degradation. Buffers containing 1mM EDTA are used to prevent this.

RNA is very sensitive to degradation by nucleases. All glass (and plastic) used must be rinsed in water containing diethylpyrocarbonate (DEPC) (2%) and autoclaved before re-use. All reagents, water and consumables used in subsequent handling must be sterile and certified RNAse free. Anion-exchange and desalting columns should be stored in sterile water/acetonitrile (1:1), except Q-res type which should be stored in water/ethanol (4:1).

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