FAQ

General information

What is GeneWorks policy on confidentiality?
What Quality Control checks does GeneWorks have in place?
I think there may be a problem with my oligo. Can I get a replacement?
What is the GeneWorks Oligo Reward Program, and how do I join?

Ordering

How can I order?
I am in New Zealand – how do I order GeneWorks oligos?
How long will it take to make my oligo(s)?
What purity oligo should I order?
Copying and Pasting sequences
Can I order an oligo with degenerate (mixed) bases?
I wish to order an oligo less than 5 bases in length. Is this possible?
How do I indicate modifications in my DNA sequence?
I want to order phosphorothioate-modified oligos for in vivo work. What purity should I select?
How do I order Probes or Phosphorothioate oligos?
Can GeneWorks make NED, VIC or PET labelled primers?
I want to develop a Probe-based assay but do not have access to design software. Can GeneWorks help?
I want to order an MGB™ Dual Labelled Probe? Can GeneWorks make this for me?
I have not received an e-mail confirmation. Should I re-submit the order?

Questions on Receipt

The amount of oligo I received is lower than I expected. Why is this?
The oligo Tm indicated on the Data Sheet does not correspond with estimates from other sources. Which one is right?
The amount of oligo in my solution does not correspond to what is printed on the label. Why is this?
I can't see my oligo / it looks different from last time.
My oligo arrived in more than one tube. How do I know how much is in each tube?
How do I resuspend and store my oligo?
How do I quantify my oligo?
Why are my oligos sometimes delayed, and what can I do to prevent this?

Technical Questions

How are oligos synthesised?
Does my oligo have a phosphate on the 5’ end?
How much full length oligo will be in my preparation?
What is the longest oligo GeneWorks can make?
I have sequenced my DNA and the oligo seems to contain errors. What is the reason for this?

 

General information

What is GeneWorks policy on confidentiality?

GeneWorks treats all details of oligos ordered as confidential to the person who ordered the oligos. Measures are in place to protect the integrity of these data and not release it to unauthorised parties. If access to another person’s oligo records is requested, we will require that person to give their consent in writing before information can be released.

What Quality Control checks does GeneWorks have in place?

Quality control is an integral part of GeneWorks ISO 9001 accreditation. GeneWorks oligos are carefully monitored during synthesis by trityl analysis (colorimetric measure of coupling efficiency). At the end of synthesis, oligos are quantified by UV spectroscopy to measure the yield and check it meets requirements. Every synthesis run is checked by denaturing polyacrylamide gel electrophoresis (PAGE) analysis of oligos prior to release for sale. This checks for the correct oligo length and that the % of full length product is within specification.

We also monitor performance of our synthesisers by Electrospray Mass Spectrometry (MS) of a proportion of our production. This analysis gives a mass accurate to within 2 Daltons, sufficient in most cases to confirm oligo integrity and base composition (sequence) of oligos.

Fluorescent primers are checked by UV-VIS spectroscopic analysis.

All GeneWorks Dual Labelled Probes are analysed after HPLC purification by PAGE and UV-VIS spectroscopy to ensure that both reporter and quencher dyes are present on the oligo.

I think there may be a problem with my oligo. Can I get a replacement?

Probably. GeneWorks' underlying philosophy is to help our customers get their experiments to work and to avoid downtime. We do this by offering technical advice and replacement where (a) this is likely to be helpful and (b) where there is a reasonable possibility the oligo is the cause of the problem. Replacing an oligo is often the easiest and quickest way to troubleshoot.

Investigating in this way also helps us add to our extensive knowledge base so we can better advise other customers in the future. In some cases we will ask you to help in supplying experimental detail or returning samples of your oligo stocks for further analysis.

What is the GeneWorks Oligo Reward Program, and how do I join?

GeneWorks values its loyal customers. We offer a range of rewards to members of our Oligo Reward Program, based on the number of oligos ordered. For more information please refer to our Rewards section.

Ordering

How can I order?

There are two ways you can order GeneWorks oligos.

Internet

The quickest and easiest method is via this website and following the order oligos links. If it is your first visit, you will need to register by following the links. Registration will result in a customer profile being generated for you which will then allow immediate ordering. On future visits, all you have to do is login and provide an order number (or credit card details) and you can enter your oligo sequences. Web ordering also allows you to look at previously ordered sequences and reorder them as required. Upon ordering, you automatically receive an e-mailed confirmation of your order with oligo details and list pricing.

For orders containing many oligos we now provide a specially designed e-mailable Excel Oligo Order Form. You can copy and paste, or type in, sequences into this spreadsheet and e-mail it to oligos@geneworks.com.au when you are ready to order.

E-mail

Orders are also accepted by arrangement to oligos@geneworks.com.au. Please make sure you provide the following information: Your Name, Delivery Address (and Invoicing Address if different), Oligo details (name, sequence, synthesis scale, purity, type and position of any modifications), Purchase Order number or full Credit Card details.

Fax (This option is no longer available)

Previously offered as an alternative ordering option was our Oligo Fax Form .

Please note that as of 1st of July 2011, GeneWorks will NO LONGER BE ACCEPTING any faxed Custom Oligonucleotide orders.

All order's must be ordered either online or by downloading the above Excel Oligo Form which can be emailed to: oligos@geneworks.com.au

I am in New Zealand – how do I order GeneWorks oligos?

GeneWorks has a web site, www.geneworks.co.nz, designed specifically for our NZ customers. Registered customers can see local pricing and delivery details, and other NZ-specific information.

How long will it take to make my oligo(s)?

The turnaround times published on this site and in our price list (from order to shipping) depend on the type of oligo ordered and what service speed is chosen (Priority or Off Peak). A table summarising our turnaround times is available on this web site, in our price list brochure and via a link on oligo order confirmation emails. These are maximum times and we often make oligos faster than this. Please note that larger orders (>20 oligos) may take additional time.

Shipping is overnight to most metropolitan VIC, TAS, NSW, ACT, NT, QLD and WA destinations and same day to most Adelaide destinations. Please see www.geneworks.co.nz for service and delivery information to New Zealand. Please enquire for other destinations.

What purity oligo should I order?

GeneWorks offers several different levels of purity. The Sequencing/PCR grade oligos are prepared to ensure that they contain no salts that would inhibit sequencing or PCR reactions. These oligos have the highest yield, and are the most stable. Transfection grade oligos have had any trace salts or organics removed and are considered more suitable for sensitive systems. Reverse phase HPLC removes failure sequences present (oligos missing bases at 5’ end), generally resulting in a final purity of 97- 99% of full length sequence.

The following table can be used as a guide to grade selection.

Grade selection guide.
Application Grade/Purity
Sequencing/PCR Grade Transfection Grade HPLC Purified
PCR, Real-time PCR Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for maximum reliability of performance in critical applications
Sequencing Generally recommended Offers no advantages over Sequencing/PCR Grade Use where highest sequencing quality is required (eg no stutters)
In vitro mutagenesis Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for oligos >50 bases and/or where missing 5’ bases in some molecules would cause problems
Transfection Not recommended. Can give rise to toxic/non-specific effects Recommended Use for oligos >50 bases where high % full length is required. Yields of phosphorothioate modified oligos can be low after HPLC.
siRNA construct Generally recommended Offers no advantages over Sequencing/PCR Grade Consider for sequences >60 bases – can reduce amount of downstream work required eg screening clones.
Cloning Recommended for oligos <50 bases or for longer oligos where process is selective for ‘full length’ oligos. Offers no advantages over Sequencing/PCR Grade Recommended for sequences >50 bases, where the process is not selective for ‘full length’ oligos. Can reduce amount of downstream work required eg screening clones.
Copying and Pasting sequences

Sometimes when you copy and paste an Oligo sequence from another applications the website ‘rejects’ it. Applications like MS Excel and MS Word sometimes have hidden characters at the end of a cell or line. This character means something to that application but is rejected by the web site.

To resolve this on the web site, go to the end of your sequence and press the delete key a few times. This should remove those hidden characters and allow the sequence to be processed.

Can I order an oligo with degenerate (mixed) bases?

Yes. There is no extra charge for this service. With the exception of the 3’ base (see below), any combination of the bases A, G, C or T can be used. The following IUB codes are used to indicate base mixtures:

R=Ag,Y=CT, M=AC, K=gT, S=gC, W=AT, H=ACT, B=gCT,
V=AgC, D=AgT, N=AgCT

Please note that the only base mixture allowed at the 3’ end is N. For other combinations multiple oligos with different 3’ bases must be ordered then combined in equimolar amounts.

I wish to order an oligo less than 5 bases in length. Is this possible?

Yes, we can make oligos this length. Please note that a ‘Set-up’ charge applies to oligos of <10 bases. Please enquire for additional information.

How do I indicate modifications in my DNA sequence?

GeneWorks uses a number code to identify modified bases in your oligo sequence. When you want to insert a modified base, refer to the relevant pull-down menu options below the oligo sequence box, select the modification of choice, and type in the appropriate number.

I want to order phosphorothioate-modified oligos for in vivo work. What purity should I select?

We recommend Transfection Grade oligos for this application. HPLC purification is problematic for phosphorothioate oligos and is normally not necessary for the length of oligos most often used in antisense work. See also What purity oligo should I order?

How do I order Probes or Phosphorothioate oligos?

Most types of Dual Labeled Probes, 3’ modified oligos and Phosphorothioate oligos can now be ordered through our web site. Alternatively, orders can be accepted by email or fax (see How Can I Order?).

Phosphorothioate modified oligos are ordered using an asterisk to indicate position of modified linkages (phosphodiester bonds) between bases. For example,

5’- A*G*G*TAGATCGGAATCGT*G*T*T -3’

indicates a 20 base oligo with 3 phosphorothioate linkages at each end.

Can GeneWorks make NED, VIC or PET labelled primers?

These are Applied Biosystems dyes that are not available to companies like GeneWorks. We recommend customers source these dyes from Applied Biosystems but consider GeneWorks as the supplier of other dyes in the set (eg FAM, TET, HEX). GeneWorks also offers TAMRA and other dyes which can be used on some genotyping instruments. We can also make oligos with dyes that have very similar characteristics to VIC.

I want to develop a Probe-based assay but do not have access to design software. Can GeneWorks help?

GeneWorks can assist in the design of Dual Labelled Fluorogenic Probes for real-time PCR prior to probe and primer synthesis in our laboratories. Using Beacon Designer 5.0 from Premier Biosoft, GeneWorks can design candidate primer and probe sequences for a given target sequence. We can usually also recommend probe sequences for use with existing primer pairs. Please contact us for more information.

I want to order an MGB™ Dual Labelled Probe? Can GeneWorks make this for me?

Due to license restrictions GeneWorks is unable to offer probes containing MGB (Minor Groove Binder). Please note that because of the Tm-raising effect of MGB, probes designed to include MGB will not work properly when synthesised without the MGB moiety.

MGB™ is a trademark of Epoch Biosciences

Does GeneWorks make RNA oligos?

GeneWorks specialises in DNA supply. For RNA synthesis we rely on the expertise of Ambion in the US, who supply a range of services designed primarily for the siRNA and miRNA researcher. Please enquire for more information on how to order Custom RNA.

How are oligos shipped and how stable are they?

GeneWorks ships oligos dry. In this form, they are stable for at least two weeks in normal shipping conditions. Unmodified oligos can usually be stored in this form for several months at room temperature. Dye labeled oligos and some other modifications are more sensitive and where possible should be stored on receipt at or below 4°C.

I have not received an e-mail confirmation. Should I re-submit the order?

No – it is unusual for there to be problem with the web site. Re-submission of the order may result in two sets of oligos being made. Please first check that the e-mail address in your registration details is correct and that there is no problem with receiving e-mail at your end. If all seems in order, please contact us immediately.

Questions on receipt

The amount of oligo I received is lower than I expected. Why is this?

The terminology for synthesis scale reflects the nominal amount of the first base on the synthesis support used. Synthesis and cleavage efficiency is not 100%, therefore the final yield can be lower than the scale ordered.

The final yield will depend on the sequence ordered, the loading of the first base on the support, efficiency of synthesis, cleavage, length and purity desired. GeneWorks guarantees a minimum amount per oligo, based on OD units. Please refer to the tables below for minimum yields.

Minimum yields
Minimum yields for a 20 base oligo
SYNTHESIS SCALE SEQ/PCR TRANSFECTION HPLC
1 OD = approx 30 µg (depends on E260, which depends on sequence and modifications)
40 nmole 5 OD 4 OD 2 OD
200 nmole 20 OD 16 OD 8 OD
1 µmole 80 OD 64 OD 32 OD
10 µmole 500 OD 400 OD 200 OD

Shorter oligos have proportionally lower minimum OD yields. Longer oligos' OD yields can sometimes be higher but this will be non-proportional and sequence dependent.

The following guide can be used to convert from OD to nmoles:

Convert from OD to nmoles
Oligo length (bases) Approx. nmoles / OD*
* These figures are sequence-dependent.
5 22.0
10 10.0
15 6.8
20 5.2
25 4.0
30 3.3
35 2.9
40 2.6
45 2.4
50 2.2
55 1.9
60 1.7
65 1.6
70 1.5
75 1.4
80 1.3
85 1.2
The oligo Tm indicated on the Data Sheet does not correspond with estimates from other sources. Which one is right?

GeneWorks uses the following formula to calculate melting temperature (Tm) of oligos:

Tm = 81.5 + (16.6 x log[Na+]) + (0.41 x %GC) - (675/oligo length)

using a default value of [Na+] = 0.1M

There are a wide variety of algorithms available for estimation of oligo ‘melting temperature’. Estimates can vary significantly due to differences in formulae and default salt concentration etc. Oligo Tm is also not a good direct indicator of annealing temperature in a PCR reaction. We advise customers to stick to one algorithm and empirically evaluate the relationship of Tm to optimal annealing temperature.

The amount of oligo in my solution does not correspond to what is printed on the label. Why is this?

There are several possible explanations. DNA pellets can occasionally be incompletely dissolved. It is wise to centrifuge the tube for a few seconds before opening (see How do I resuspend and store my oligo?) to make sure the pellet is at the bottom of the tube when buffer is added. For critical experiments it is a good practice to measure the concentration of oligo present after dissolution (see How do I quantify my oligo?).

I can't see my oligo / it looks different from last time.

Our custom oligos are provided as desiccated pellets. It may have dried to a translucent pellet, in which case it is difficult to see. Drying is a variable process, and occasionally the pellet may look fluffy rather than clear. This is normal, and will not affect the performance of your oligo.

Occasionally oligos may retain minute amounts of fluorescent dyes from previous syntheses which can add a slight colouration to dry oligos. This will not interfere with use of the oligo in most applications.

My oligo arrived in more than one tube. How do I know how much is in each tube?

Oligos are sometimes supplied in more than one tube. In such cases, the amount printed on the label and data sheet is the quantity per tube, not the total amount provided. The DNA is usually split equally between the tubes.

How do I resuspend and store my oligo?

Collect the DNA in the bottom of the tube by pulsing in a microcentrifuge. Open tube and add the desired amount of buffer. Allow to rehydrate for a few minutes with intermittent vortexing before quantitation. GeneWorks recommends that oligos be resuspended in 10mM Tris. pH 8.0, TE or nuclease-free water. Unmodified oligos can be stored for at least 6 months at -20°C, however we suggest that multiple freeze/thaw cycles be avoided. Aliquoting stock solutions after resuspension and before freezing will allow small amounts to be thawed at once.

Some modified oligos are less stable and should be used within 3 months for ideal performance. This applies in particular to Cy3 and Cy5 labeled oligos. All dye-labeled oligos should be stored in the dark.

If two or more tubes of each oligo are supplied, a single tube should be resuspended and other tubes stored as a desiccated pellet.

This and other useful information is available on GeneWorks’ laminated ‘Working with Oligos’ card.

How do I quantify my oligo?

Accurate quantification can be done by determining the absorbance at 260 nm of a diluted aliquot of the oligo. To do this, first dilute an aliquot of the resuspended oligo in water to a final volume of 1000 µl. Calculate your dilution factor from this step. For example, if 50 µl was diluted, the dilution factor would be 1000/50 = 20. Vortex. Read the absorbance of the dilution at 260 nm. To calculate the concentration of the oligo from the absorbance reading, multiply the absorbance by the dilution factor and then multiply again by the weight per OD (roughly 30 µg/ml).

Approx. concentration (µg/ml) = A260 x 30 µg/ml x dilution factor

For a more accurate quantification the Extinction Coefficient can be used:

Concentration (M) = A260 / E260 (L/mole.cm)

where A260 is measured in a 1cm path length cuvette.

Why are my oligos sometimes delayed, and what can I do to prevent this?

As with any system, problems occasionally occur which affect production of oligos. This can include ‘yield failures’ or oligos that do not pass our pre-sale Quality Control checks (see What Quality Control checks does GeneWorks have in place?).

In the event of delays beyond our published turnaround times, we contact the customer to advise them of the situation. In some cases it is possible to arrange shipment of other oligos on the order while the others are being remade.

Our dispatch times are based on an order of 20 oligos, although we do endeavor to complete all orders as quickly as possible. We will contact you if it is not possible to complete a large order in the published times and ask you for the priority of your sequences.

Orders can sometimes be slowed by inclusion of ‘slow’ oligos – ie oligos with a longer turnaround time than the others. In such cases we advise to split your orders into oligos of similar turnaround time. If you need your oligos very urgently we can often help by pre-shipping the oligos in an order that are ready.

Technical questions

How are oligos synthesised?

GeneWorks oligos are made using highly efficient synthesisers from leading manufacturers. Oligo synthesis is done on a support which contains the 3’ base (A, G, C or T). Oligos are synthesised in the 3’ to 5’ direction in a ‘one base at a time’ reaction which is normally >99% efficient. Any molecules with failed base couplings are ‘capped’ to prevent extension and resultant missed bases in the oligo sequence. Because of the 3’ to 5’ direction of synthesis, any capped ‘failure’ sequences are missing bases towards the 5’ end.

Does my oligo have a phosphate on the 5’ end?

No. Our custom oligos do not have a 5’ –OH group. We can add a phosphate for you if you request it when ordering (5’ phosphate modification) for a small additional charge.

How much full length oligo will be in my preparation?

The % of full length oligo in a preparation depends on the length of the oligo and whether the oligo is purified. The sequence itself can also affect synthesis efficiency.

GeneWorks uses state of the art instrumentation and the highest possible quality reagents to maximise synthesis efficiency. Measurement of our oligos indicates average coupling efficiency is approximately 99%. The graph below shows the % full length product for oligos of various lengths that would result from 99% coupling efficiency.

Graph - Purity at 99 percent Efficiency
What is the longest oligo GeneWorks can make?

GeneWorks can attempt to make oligos up to 110 bases in length.

Most sequences can be made with reasonable yield and purity however some oligo sequences can be problematic to synthesise and this is particularly so for long oligos. The longer the oligo, the more likely difficulties will be experienced and the % full length product may become very low (see How much full length oligo will be in my preparation?). As a result, GeneWorks recommends HPLC purification for very long oligos to remove failure sequences.

If an oligo synthesis still fails after the second attempt, we will contact you to discuss available options.

I have sequenced my DNA and the oligo seems to contain errors. What is the reason for this?

The cause of sequence discrepancies in cloned oligos (annealed oligos or PCR products) is almost certainly not oligo sequence error.

Oligo sequences are entered into our database (without human intervention in the case of web or GeneWorks spreadsheet orders). Sequences are then sent electronically to both synthesiser and Data Sheet/Label printer. The chance of sequence discrepancy between Data Sheet and (full length) oligo sequence is therefore negligible.

Extensive analysis of feedback from customers, and Mass Spectrometry testing of oligos which have given rise to sequence discrepancies, suggests that most reports of deletions or substitutions are due to unpredicted annealing behaviour of oligos, effects of polymerases or ligation enzymes, or the result of sequence change caused by biological selection pressure against the newly introduced sequence.

As a result, we therefore advise customers to check several clones from each ligation/transformation. In cases where sequence discrepancy is detected, GeneWorks can make a replacement synthesis by arrangement. Customers should be aware that resynthesis of an oligo sequence often gives rise to similar results.

 

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