Frequently Asked Questions

Frequently Asked Questions

FISH Probe Storage Temperature Range

Empire Genomics FISH probes are shipped at ambient temperatures. Stability data supports the conclusion that probes are unaffected after storing the probes for six (6) months at room temperature.

As noted on the probe label, the ideal storage temperature is -20°C. However, these probes are stable at a wide temperature range ( -20°C, +/- 10°C) with no discernible effects on probe performance.

Multiple freeze/thaw cycles are not recommended because this destabilizes the probe and negatively affects performance.

Download a copy of this advisory.

Vector pBACe3.6 Information/Map

In the course of our initial work with PAC libraries and PAC vectors, we obtained feedback from several sequencing centers who considered the costs of sequencing PAC versus BAC clones. A disadvantage was the size of the PAC vector (16 kb left in recombinants) as compared to the size of the BAC vector (7 to 8 kb, depending on the vector variant). For the use of BACs versus PACs with inserts in the range of 100-150 kb, this means that a larger fraction of the clones in shotgun sequence libraries (M13 or pUC) represents the PAC/BAC vector sequence. This results in a few percent increase in the cost per base pair of sequence reads.

We have, therefore, improved the BAC vector (Shizuya H. et. al. 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797) to include some of the retrofitting options included in our pPAC4 vector We also included the sacBII gene for use as a positive-selection marker to favor recombinant clones over non-insert background colonies. The sacBII gene from Bacillus amyloliquefaciens was derived from Nat Sternberg's P1 vector (Pierce et al. 1992, Proc. Natl. Acad. Sci. USA 89:2056-2060). The toxicity of the sacBII gene is a result of the conversion of fructose (derived from sacharose) into polyfructose (levan), which is toxic in E.coli. The cloning vector and recombinant clones do NOT express the sacBII gene and hence are sucrose-resistant. Undesirable clones derived from the deleted cloning vector (without the "green" stuffer fragment) are sucrose-sensitive and, hence, do not form colonies on sucrose-containing media.



The new vector has been named pBACe3.6. Specifically, we maintained the wildtype loxP site, added an additional mutant loxP511 site, a site for the intron encoded nuclease PI-SceI and the Tn7att site.

The presence of this pUC plasmid serves dual functions: high copy number of the vector for preparing large quantities and appropriate disruption of the sacBII gene to increase viability of the vector containing strain. In addition to the BamHI site, 5 additional sites can be used for preparing BAC libraries: SacI, SacII, MluI, EcoRI, and AvaIII. The EcoRI site is particularly important in view of the use of this site in the RARE cleavage procedure, thus opening the possibility for selective cloning of similar fragments from different DNA donors.

Reference: Frengen, E. et al. (1999) A Modular, Positive Selection Bacterial Artificial Chromosome Vector with Multiple Cloning Sites. Genomics 58: 250-253. Reprints available upon request.

pBACe3.6 clones have chloramphenicol antibiotic resistance. Clones should be grown in LB containing 12.5 ug chloramphenicol/ml.

What are the differences between probe products?

PROBE DETAILS


DNA Probes Custom Probes Gene Specific Probes/
Control Probes
ASR Probes*
Analytic Quantification
Signal Intensity*
DNA Sequencing
DNA Localization
FDA Registered
Turnaround Time ≥ 3 days ≥ 7 days 24 Hours** 24 Hours
Minimum Volume 40L/800ng ≥ 5 Tests 10/20 Tests 20 Tests
Pricing $150 $220 $300 As little as $60/Test

*For Custom Probes, signal intensity is tested in batch QC

** Based on availability

What happens when a probe fails QC?

If the probe fails one or more steps of our QC it will automatically be put back into production and the customer will be notified. If the probe fails a second time it will need to be replaced by the customer.

What labeling kit should I use?

The labeling kit we would recommend for your probes would be as follows:
Nick Translation Kit 50 Reactions
From Abbott Molecular
SKU: 07J00-001

For DNA purification we recommend this kit:
Agencourt CosMCPrep - High/Low Copy Fosmid and BAC Purification
Item No: A29174

Where can I find a Material Safety Data Sheet?

You can find the Material Safety Data Sheet for Labeled BAC clones by clicking the following link: Material_Safety_Data_Sheet.pdf

My package arrived with no dry ice, are my labeled clones still useable?

It is possible that when your package arrives, all of the dry ice will have evaporated. Our clones are stable at room temperature and are therefore still useable.

Can you create custom probe panels?

Yes. We can create 2, 3 and 4 probe cocktail panels upon request. Pricing is dependent upon probe volume and dyes used. For more information please contact us at:

700 Michigan Avenue
Suite 200
Buffalo, New York 14203
USA
info@empiregenomics.com
1-800-715-5880
1-716-856-3873

There is poor metaphase morphology

Repeat the assay on a new specimen using one of the following:
- Decrease the melt temperature 2C
- Decrease the melt time until morphology becomes adequate

Pretreat the slides:
1. Prepare a solution of 2X SSC/1% paraformaldehyde
2. Immerse the slide for 1 minute in 2X SSC/1% paraformaldehyde
3. Immerse the slide in purified water several times
4. Dehydrate slides through 1 minute series of ethanol rinses (70%, 85%, 100%)
5. Air dry the slides and continue preparing slide for Codenaturation

If poor morphology continues, modify use of denaturation/hybridization system:
1. Prepare 280 uL 70% formamide/2X SSC denaturation solution (196 uL formamide/28 uL 2X SSC/56 uL purified water
2. Run HYBrite or ThermoBrite Hold Temp program set at 73C
3. Place 10uL of 70% formamide/2X SSC denaturation solution on each target area and cover slip
4. Upon HyBrite or ThermoBrite reaching 73C, place slides on heating surface and close the cover
5. After 3 minutes, remove the slides
6. Remove the cover slip
7. Continue in dehydration step of non-codenaturation assay process

There is speckling due to an abnormal diffuse signal

Repeat hybridization using one of the following:
- Decrease the melt temperature by 2°C
- Decrease the melt time until signal intensity becomes acceptable

The probe appears dim

Repeat the assay on a new specimen using one of the following:
- Increase the hybridization time
- Increase the melt temperature as needed until the signal becomes adequate
- Wash the slide with 0.4X SSC/0.3% NP-40 at 70-73°C

Codenaturation led to cross-hybridization

Repeat the assay on a new specimen using one of the following:
- Increase the temperature of 0.4X SSC/0.3% NP-40 by 2°C; continue to increase the temperature as needed
- Decrease the melt temperature by 2°C

The counterstain was too bright or weak

Did the counterstain appear too weak?
-Remove cover slip and Immerse slides in 2X SSC/0.1% NP-40 at ambient temperature for 5 minutes; agitate occasionally. -Dehydrate slide through series of ethanol rinses. Air dry and reapply new counterstain

Was the wrong concentration of counterstain used?
-If too bright, dilute the counterstain in antifade solution before applying

Was the counterstain too old or exposed to light for an extended period?
-Store counterstain at -20°C, protected from light
-Do not use counterstain past its expiration date

There is low signal specificity

Were the probes diluted inappropriately?
-Ensure the probe mixture was made according to protocol specifications

Were the conditions inappropriate for hybridization?
-Ensure incubator is at the proper temperature of 37°C
-Check that the hybridization buffer was added to the probe mixture and in the adequate amount

Was the wash temperature too low?
-To maintain the wash temperature, place no more than four slides in the wash at a time and ensure temperature of the wash solution is correct prior to washing another set

Was the wash solution stringency too low?
-Ensure wash solutions are made according to protocol
-The lower the concentration of salt, the higher the concentration of formamide and NP-40, and subsequently, the more stringent the wash

There was a weak signal or no signal

Was the specimen slide not adequately denatured?
-Ensure temperature of denaturation solution is 73°C prior to immersing slide
-Increase temperature of denaturation solution to 74°C
-Increase the time slide is immersed in denaturation solution by 2-4 minutes

Was the specimen slide not adequately prepared for FISH?
-Contact Empire Genomics Technical Support
-Refer to FISH probe protocol

Were the specimen slides improperly aged after dropping the specimen?
-Age slides at ambient temperature for 24 hours prior to FISH

Were the specimen slides not dried thoroughly enough prior to immersion?
-Dehydrate slides through a series of 1 minute ethanol rinses (70%, 80%, and 100%)

Was specimen GTG-banded?
-Prepare fresh specimen slides
-Using trypsin-Giemsa banded specimens for FISH may require adjustments in banding and/or hybridization protocol- For more information contact Empire Genomics Technical Support

Was the probe not added?
-Prepare a new probe mixture, allowing enough time for probe to thaw. Vortex or pipette reagents to mix; centrifuge briefly. Pipette probe slowly

Was one or all of the components (probe, hybridization buffer, probe mixture) not mixed well prior to use?
-Use pipet to mix or vortex reagents; briefly centrifuge

Were the probes not properly diluted for hybridization?
-Maintain proper ratio of probe mix by using the volumes stated in the probe preparation procedure add 7 L hybridization buffer, 1 L CEN probe to 2 L of probe
-Ensure the pipette is calibrated. Allow hybridization buffer to thaw completely and reach ambient temperature prior to use; pipette slowly

Was the probe not adequately denatured?
-Ensure temperature of water bath used to denature the probe mix is 73°C
-Denature probe mixture for 5 minutes
-Plan so the probe is applied directly after removal of slides from the 100% ethanol solution (ensure ethanol has completely evaporated prior to application)
-Remove tube containing probe mix from the water bath and place immediately onto 45-50°C slide warmer.
-Keep tube on warmer while pipetting the probe onto the slide
-Note: only process as many slides as you can while maintaining proper temperatures and times outlined

Was the probe mix dried too thoroughly on specimen slide?
-Immediately place the cover slip over the target area after application of probe mix
-Washing the hybridization, remove cover slips from slides one at a time. Immerse each slide immediately into wash solution upon removal of slip before removing the next

Did you see air bubbles trapped under the cover slip during hybridization?
-In applying cover slip, first touch the surface of the probe mixture. Place the slide with cover slip down on a blotter, gently press out visible bubbles

Were the hybridization conditions not optimal?
-Ensure that the stated time and temperatures in protocol are strictly followed
-Ensure temperature of the incubator is 37°C
-Seal cover slip with rubber cement to ensure there are no gaps
-Increase the hybridization time

Were the wash conditions or solutions used incorrect?
-Adhere to protocol in making wash solutions and setting temperatures
-Ensure the thermometers and pH meters used are calibrated properly
-Before immersing slides in wash solution, remove cover slips


Were the probes or specimen slides stored improperly?
-Store undiluted probe at -20°C in the dark
-Store non-hybridized slides desiccated at -20°C (for extended periods) or at ambient temperature (for shorter periods)
-Store hybridized slides at -20°C in dark for up to 6 months

Was the wrong filter set used to view hybridization?
-Probe signals may appear fainter if viewed through multi-bandpass set; use correct filter for flourophores

Was the microscope configuration or objective not adequate to view FISH results? Are the filters damaged?
-Contact your microscope manufacturer

The FISH assay resulted in a high slide background

Was slide cleaned properly prior to sample preparation?
-Immerse slide in ethanol, dry using lint-free paper

Were the Specimen slides used too soon prior to denaturation?
-Ensure slides are aged at least 24 hours at ambient temperature

Could there have been too much cellular debris on the sample?
-Wash cell pellet with fresh fixative three times prior to dropping slides

Were the metaphase spreads aged by baking, or do they contain cytoplasm?
-Increase time the slide is immersed in the denaturation solution to 10 minutes

Was the slide inadequately washed following hybridization?
-Ensure wash solutions were made according to procedures outlined by the package insert
-Make sure the wash solution is at the proper PH and temperature, remove cover slip and repeat the wash procedure
-Increase immerse time to 4 minutes in the 73°C 0.4XSSC/0.3% NP-40 wash

Were the wash solutions used too long or improperly stored?
-Ensure wash solutions containing formamide are properly stored at 4°C
-Discard these solutions after 7 days or after frequent use; discard all other solutions after 1 day
-Ensure the pH of formamide solutions are pH 7.0-8.0

Was the hybridization viewed using long bandpass filters?
- Reduce background light by switching to filters with smaller bandwidths or to multi-bandpass filters

The FISH assay resulted in distorted chromosome morphology

Did the slides dry too quickly during preparation?
-Increase the humidity used when dropping slides, or increase the temperature of the water bath.
-Decrease slide warmer temperature during preparation of samples
-Let slides dry overnight; age for at least 24 hours at room temperature
-Do not bake slides at high temperature

Was the specimen over-denatured?
-Adhere to directions on package insert to make denaturation solution
-Prior to immersing the slide, ensure temperature of the solution is at 73°C, decrease temperature to 72°C
-Decrease the time of slide denaturation process by 1-3 minutes

How do Empire Genomics dyes compare to dyes offered by other vendors?

This is how other vendors' dyes compare to EG:


Color



Label Description



Ex. Max



Em. Max



Aqua



DEAC-dUTP



433 nm



480 nm



Green



5-Fluorescein dUTP



497 nm



524 nm



Green



Cyanine-3 dUTP



509 nm



538 nm



Gold



5(6)-Carboxyrhodamine 6G dUTP



530 nm



555 nm



Orange



5-TAMRA dUTP



559 nm



588 nm



Red



5-ROX dUTP



592 nm



612 nm



Red



Cyanine-5 dUTP



655 nm



675 nm




 

Do you have a guarantee for the shelf life of your FISH probes?

We provide a 2 year guarantee on all FISH probes from the date of manufacturing.

What dye colors are available for BAC probe labeling?

The following dyes are available when ordering our Custom Labeled BAC Probes:
Color Label Description Ex. Max Em. Max Ext. Coeff.
Aqua DEAC-dUTP 431 nm 480 nm 50000
Green 5-Fluorescein dUTP 496 nm 520 nm 85000
Gold 5(6)-Carboxyrhodamine 6G dUTP 525 nm 551 nm 92000
Orange 5-TAMRA dUTP 552 nm 576 nm 60000
Red 5-ROX dUTP 580 nm 603 nm 75000

What dye colors are available for BAC probe labeling?

The following dyes are available when ordering our Custom Labeled BAC Probes:
Color Label Description Ex. Max Em. Max Ext. Coeff.
Aqua DEAC-dUTP 431 nm 480 nm 50000
Green 5-Fluorescein dUTP 496 nm 520 nm 85000
Gold 5(6)-Carboxyrhodamine 6G dUTP 525 nm 551 nm 92000
Orange 5-TAMRA dUTP 552 nm 576 nm 60000
Red 5-ROX dUTP 580 nm 603 nm 75000

Is there an available protocol or instruction guide for your Custom Labeled BAC Probes?

Yes. Please download the PDF version of our Labeled FISH Probe Instruction Guide. If you have any additional questions, please feel free to contact us at info@empiregenomics.com.

How many hybridizations are possible with each probe order?

Each labeled FISH probe order enables you to perform 5 hybridization experiments. For more information, please review the Labeled FISH Probe Instruction Guide.

How do I place an order for labeled BAC probes?

If you already know the clone name (ie: RP11-79G24) of the BAC you would like to have fluorescently labeled, please go directly to our labeled FISH probe ordering page. If you would like to search for a clone based on a gene or chromosomal region of interest, visit CloneCentral - our new BAC clone search utility.

What is the turnaround time for labeled BAC probes for FISH?

You can receive your order in as quick as 7 days. Please note: Orders are currently processed once a week and must be received by Monday at 12 noon EST for quickest turnaround time. Average turnaround time is 10-14 days.


How much DNA comes with each labeled RP11 BAC probe?

All labeled BAC probe orders come in a 5-test kit format which includes a total quantity of 1ug of DNA. Click here to learn more about our Custom Labeled FISH probes.

How do I prepare the RNA sample for mailing?

An easy and safe way to ship total RNA samples is in RNAstable (from Biomatrica, http://www.biomatrica.com/rnastable.php). The product has been tested and found to be excellent for storage and shipping of RNA at room temperature when manufacturer instructions are carefully followed. After RNA is dried down in RNAstable, the sample must be enclosed in an airtight container or pouch with desiccant added to protect the RNA from ambient humidity. Your sample can be shipped at room temperature and for a much lower cost than shipping on dry ice. If the RNA does not have a preservative, please ship on dry ice and pack with adequate dry ice to keep RNA frozen for several days. International shipping routinely takes a minimum of two day and any delays in shipping will result in possible loss of sample integrity if an adequate amount of dry ice is not used. All shipments on dry ice should be mailed on a Monday or Tuesday to avoid having delayed shipments arrive on the weekend. The Empire Genomics facility does not receive samples over the weekend or during holidays.


Who can I contact if I have questions?

You can contact info@empiregenomics.com with your questions. Alternatively, we welcome your phone call at 716-856-3873 or toll free in the US & Canada at 1-800-715-5880.

Where can I get more information about the Agilent platform?

Agilents websites (www.chem.agilent.com) have extensive technical support sections including FAQs on all steps of the protocols used, arrays available and a literature library section with papers, technical notes, and posters.


What software is used for gene expression data analysis?

GeneSpring GX is a powerful visualization and analysis solution designed for use with gene expression data. The software provides advanced statistical tools, a range of visualization displays (scatterplots, pathway diagrams, and more), and data quality evaluation. Visit Agilents website for more information about GeneSpring GX.

Are there controls spotted on the Agilent arrays?

Yes. Some act as negative controls and there is also a set of positive controls that are spiked into the hybridization cocktail.

What is the length of the oligos on the Agilent arrays?

They are 60 nucleotides long.

What steps are taken to ensure the least amount of technical variability?

All of our technicians are experienced, highly trained individuals. Whenever possible, the same technician will perform all experiments in a project. Whenever possible we will use reagents from the same lot, arrays from the same print batch, and use the same equipment (hyb oven, scanners, etc) to reduce technical variability.


How long will it take to get results back?

A typical experiment of 6-12 arrays takes about 1 week (including the basic data analysis service) from the time we receive the RNA and verify its integrity on the Agilent Bioanalyzer.


What are the advantages and disadvantages of 1-color and 2-color hybridizations?

The decision to use 1-color or 2-color is mainly a factor of the experimental design and the research question asked. 1-color experiments are generally less expensive as less labeling reagents are required. In addition, a control RNA is not required on each array, often preserving precious sample. 2-color experiments generate results in a ratio format rather than absolute numbers. Technical variation between arrays is automatically accounted for since a reference RNA is used for each co-hybridization.


Do you check for appropriate dye incorporation when generating labeled probes?

Yes, all fluorescently labeled probes are quality control checked on a Nanodrop ND-1000 spectrophotometer for appropriate incorporation before proceeding with hybridization.

What if my total RNA sample appears to be slightly degraded on the Agilent Bioanalyzer? Can I proceed with labeling?

All RNA samples should have a Bioanalyzer 2100 rRNA [28S/18S] ratio > 1.5, RIN >7 to pass QC. Occasionally, researchers will carry on with an experiment even if the RNA is slightly degraded knowing that this is the highest quality RNA they can isolate. Empire Genomics will notify you prior to starting the gene expression process if the RNA fails QC.

Why do you analyze my RNA on the Bioanalyzer?

Assessing the quality of your RNA sample before proceeding with labeling and hybridization can save a lot of time and money. It can also reduce the time spent troubleshooting if something goes wrong at the next step. The Agilent Bioanalyzer is a convenient replacement for standard denaturing agarose gels; it is much faster and requires only 1µL at a concentration of ~100 ng/µL for analysis. The Bioanalyzer uses capillary electrophoresis (lab-on-a-chip technology) to move the sample through a gel matrix. The resultant electropherogram provides the user with a detailed trace of each sample and a metric of RNA quality (RIN).


Do you offer gene expression services for low RNA yields?

We can amplify 50-100 ng of total RNA using the Low RNA Input Linear Amplification Kit from Agilent Technologies. This method requires only one round of amplification and produces either cyanine-labeled cRNA (anti-sense) or cDNA (sense) from total RNA, depending on RNA source.

How much total RNA is required for gene expression profiling?

We require a minimum of 2µg of total RNA (50-250ng/µL, treated with DNase I, 260/280: 1.9-2.1) per labeling reaction. This amount will also allow us to perform quality control analysis using the Agilent Bioanalyzer.

What is the difference between a technical replicate and a biological replicate? And which type is best?

A technical replicate involves the multiple labeling or reciprocal labeling of the same RNA sample. The purpose of a technical replicate is to control for technical variability within an experiment (array to array variation, reagent variation, dye incorporation, etc.). A well run experiment should have minimal variation thus minimizing the need for technical replicates. A biological replicate involves isolating RNA independently from replicate sources (multiple cell lines, multiple biopsies, multiple patients, etc.). The purpose of a biological replicate is to control for biological diversity. Biological replicates are often more telling, and for this reason are better than technical replicates, however, biological replicates are often more difficult to obtain.


Should I request a dye-flip labeling?

Dye-flip (reciprocal) labeling is recommended for expression analysis when fluorescently labeling cDNA as the dyes incorporate with different efficiencies.


How many replicate experiments should I do?

We recommend that you consult with a biostatistician to determine the proper number of replicates. Typically, three replicate experiments are required for small sample sizes if data analysis is to be performed with even minimal confidence. Larger sample sizes may only require a single replicate experiment.

Can I talk to someone about experimental design?

Yes, you can contact info@empiregenomics.com with your specific questions. You can also call us at 716-856-3873 or toll free in Canada/U.S.A. at 1-800-715-5880.


Is any special paperwork required for shipping sample to you from outside the US?

Yes. If you are shipping RNA/DNA to Empire Genomics from outside of the US, you are required to include Customs documentation with your shipment. To prepare this documentation, we require the following information from you: your name, mailing address with postal code, e-mail address, phone number, name of courier you are using (i.e. FedEx), the number of packages being sent and the approximate weight/size; and a description of what you are sending (non-hazardous, non-toxic, non-infectious RNA/DNA only; please specify the organism the RNA/DNA is from and the number of tubes you are sending with approximate volume of each). Please indicate the samples as having zero value. If you are shipping within the US, no such documentation is required.


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