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FAQS
Quantitative PCR assays
In our facility most people are using Sybr-green assays to perform quantitation by PCR. This assay uses a double stranded DNA binding dye which fluoresces only in the presence of dsDNA. The Sybr-green assay is economical and sensitive, but requires highly specific primers. If non-specific PCR products are produced, quantitation will not be possible with Sybr-green because the dye will bind to any product (as well as primer di-mers).
Alternately, one can use Taqman assays in which a third primer is used in addition to the traditional PCR primer pair. This third primer, called a probe, is labeled with both a fluorescent molecule and a quencher molecule. It binds to the target sequence between the two PCR primers. As the reaction proceeds, the exonuclease activity of Taq enzyme cleaves the probe, liberating the fluor and quencher molecules. Since the fluor and quencher are no longer in close proximity fluorescence is no longer quenched, and the fluorescent signal is then collected by the RealTime instrument. The advantage to this type of assay is that more than one gene may be monitored in a single reaction (i.e. fluors with different emission wavelengths may be used together). Multicolor detection allows a control gene (usually a constitutively expressed gene whose expression does not vary in response to environment) to be amplified along with the gene of interest, simplifying the analysis phase of the experiment.
The drawback to using multicolor detection is that it can be expensive. Our facility usually recommends that studies involving large numbers of experimental data points for only a few genes will benefit from Taqman assays. On the other hand, studies in which a large number of genes must be validated among a small number of RNA samples will be more economical when using Sybr-green detection.
Below are some additional points for those considering quantitative PCR.
You will need special plasticware with optical quality covers. These are available from several distributors such as Applied Biosystems, Axygen, and Bio Rad. The Applied Biosystems plasticware is designed to be most compatible with the ABI 7000.
The following plasticware is recommended:
96-well optical plates (Fisher Scientific #AB-1400)
384-well optical plates (Fisher Scientific #AB-1310)
optical adhesive covers (Fisher Scientific #AB-1170)
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optical tube strips (Applied Biosystems cat#4316567)
optical strip caps (Applied Biosystems cat#4323032)
Master mixes/enzymes
When doing quantitative PCR, the first step is to make cDNA. We have found that the Superscript III reverse transcriptase (Invitrogen cat#18080-044) works well. There are also first strand synthesis kits that have primers (oligo dT and/or random primers) already in the master mix, such as the Bio-Rad iScript cDNA synthesis kit (cat #170-8890), among others. If using gene-specific primers to prime the cDNA reaction, as well as the PCR reaction, a one-step kit may be used. This is a convenient approach because all of the reactions may be done in the same tube. No purification of the cDNA template is necessary prior to PCR. Please contact the for more information on these kits.
When using the ABI 7300 or 7900 instrument, it will be necessary to use a qPCR master mix that contains a ROX passive standard. Using a ROX fluorescent standard will correct for variability in excitation light source levels from well to well. There are several such master mixes. Below are just a few which can be used with Sybr-green detection assays. Feel free to contact the Genomic Analysis and Technology Core for information on master mixes for use with Taqman probe assays.
The following master mixes are recommended:
Sybr-Green PCR Master Mix (Applied Biosystems cat #4309155)
Bio-Rad iTaq Sybr green supermix with ROX (Bio-Rad cat #170-8850)
Sybr Green primer design
Due to the fact that Sybr-green binds to all double stranded DNA, it is extremely important to have a good primer pair which does not form dimers. It is best to avoid using primer-pairs that create dimer structures with DG higher than 6kcal. There are several good web based primer design programs. Our facility recommends Primer3 for its simplicity of use. Commercially available programs such as Oligo work very well also.
Dual labeled probe design
The Genomic Analysis and Technology Core facility has Applied Biosystems™ Primer Express software for those interested in Taqman probe design. This software works well. However, the Assays-by-design service from ABI can save time and effort. For a fee, ABI can design primers and probes for your gene of interest. They will functionally test the reactions, and send you a master mix ready for use. Feel free to contact our facility for further information.
Normalizion techniques
Variability in RNA template concentrations will make quantitation by PCR inaccurate. There are two common ways to control for variations in total RNA levels in each RT reaction. The first is to use a control gene like B-actin or GAPDH to act as a loading control. Sometimes, the use of a housekeeping gene is not possible, in which case RNA concentrations need to be standardized. The most sensitive and accurate means of measuring RNA is with RiboGreen reagent from Molecular Probes. You will need a fluorometer to use this system. Also, a UV spectrophotometer may be used.
Data replication
It is always good practice to replicate data points at least three times for any experiment. The sensitivity of qPCR makes this practice a necessity. Inaccuracies in pipetting and RNA quantitation will invalidate quantitative PCR results if not compensated for. When replicates are performed, standard deviations will reveal the reproducibility of the data.
PCR efficiency
It is VERY important to know the efficiency of your quantitative PCR reaction. If the reaction is not proceeding at 100% efficiency, then there will not be a doubling of PCR product in each cycle. Fold change calculations rely on the assumption that there is a doubling of product per cycle. Two reactions may be directly compared to each other, if they are operating at the same efficiency (preferably above 90%). However, if the control and gene of interest are amplifying with different efficiencies it is not recommended that they be directly compared.
There is a mathematical technique for correcting for PCR amplicons of different efficiencies presented by M. W. Pfaffl (Nucleic Acids Research, 2001, Vol. 29, No. 9 e45). Please contact the Genomic Analysis and Technology Core facility if you need further information on the subject. |
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How do I begin using the Genomic Analysis and Technology Core sequencing facility?
- University of Arizona FRS accounts may be used to pay for services rendered. The account must be current, and contain enough funds to cover the charges.
- Off-campus users must create a P.O. with the Arizona Research Labs business office. Please email Bob Quiroz to begin setting up a P.O. Please allow sufficient time for processing of P.O. information before using the Genomic Analysis and Technology Core services.
- The next step is to create a profile in the On Core data management system. There are two separate systems for the low volume service and high volume service.
- Once your profile has been created, you can begin submitting sample information. Please be sure to complete the on line work request before bringing the templates to the lab in Life Sciences South Room 203.
Where do I take my DNA to be sequenced?
- Our facility is located in the Thomas W. Keating BioResearch Building Room 124. Please give your templates to one of the sequencing staff members. They will check in your templates on line.
- Users at the south campus may drop off their templates in the refrigerator in Life Sciences South Room 205. There is a daily pickup from that location.
- All out of town users can mail their samples tothe following address:
Genomic Analysis and Technology Core - DNA Sequencing
Arizona Research Laboratories - Division of Biotechnology
Thomas W. Keating BioResearch Building
1657 E Helen St. #124
Tucson, AZ 85721-0240
How do I prepare my templates for sequencing on the low volume service?
If you do not find answers here, please do not hesitate to contact us directly at 520-621-9184 or
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Submitting DNA Templates:
Submit all samples in 1.5ml tubes. Write name of template exactly as it appears on the web site work order. On the side of the tube, write your name and the tracking number from the web site. Please note in the comments section of your work order if you anticipate difficult template (e.g. GC rich). If the template is to be used for more than one sequencing reaction, submit only one tube with enough DNA for all of the reactions.
Required Volumes for Submission:
- Template- 8µL/reaction
- Primer- 5µL/reaction
Sample Preparation for PCR products:
The most important factors are concentration and purity . We strongly recommend that all PCR products be prepared with commercially available PCR cleanup kits to remove excess primers, nucleotides, and buffers. The most commonly used PCR purification kit among our users is Qiagen's QIAquick PCR system.
Important! Do not use buffers containing EDTA (such as T.E.) to resuspend your templates. This will cause your sequencing reaction to fail! We recommend submitting samples in sterile ultrapure (MilliQ) water.
What are the ideal concentrations and quantities for templates and primers?
Template
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Concentration
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*total ng/ reaction
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| Plasmids (up to 10 KB including insert) |
200ng/µL |
500 ng |
| Plasmids (over 10 KB including insert) |
200-400 ng/µL |
500-1000 ng |
| BAC DNA |
1-2 µg/µL |
5 ng |
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Cosmids, Phage DNA
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1µg/µL |
2.5 ng |
PCR: Optimum concentration depends on length . Use the formula: Length (Base pairs) x 0.02 = Concentration (in ng/µL )
* Templates at optimal concentration use a standard 2.5ul of template per reaction. If template concentration is lower than ideal concentrations, we can use up to 8ul of template. When you submit your sample online, please enter correct concentrations. Our computer will then calculate how much template will be optimal in your reaction.
Primers: 3-5pmol/µL (3-5µM)
Note: for a 20-mer primer this is 20ng/ul.
How do I prepare my samples for sequencing on the high volume service?
- Templates are brought to the facility in 96-well format.
- If using more than one primer on the plate, please provide a plate with primers in the wells corresponding to the proper template locations.
- DNA must be of high purity, and consistent concentration for best results. The 3730xl sequencer is very sensitive to salts.
- Users wishing to use the facility's automated sample cleanup, quantification, and normalization services should submit their plates to those services before sequencing.
- Optimum concentration for PCR products is 10ng/ul & for plasmids 50ng/ul. Please supply at least 8ul of template for each reaction to be performed.
- Optimum primer concentration 3 pmoles/ul. Please supply at least 4ul of primer for each reaction to be performed.
What instrument do you use to analyze the sequencing products?
*Equipment and description*
How long will it take to get my results?
- Average turn around time on the low volume sequencing service is 3 business days.
- For the high volume service, expect results in 2 to 3 days.
How do I view my sequences?
The files that our automated sequencers generate require specialized software in order to be viewed. As you might expect, these viewers are platform specific. Please see the notes at the bottom of this page on how to configure your computer's file associations so that the files will open when you click on them
There are several free viewers that are available to look at the electropherograms. Below are a few of the options. These programs are for viewing individual sequence files. If you need assistance with large scale sequencing data management, please feel free to contact us for assistance or recommendations.
Alternately, a text file containing only the sequence of nucleotides can be downloaded from our server. The text file can then be imported into your favorite sequence analysis package for analysis or alignment. Be aware that the Genomic Analysis and Technology Core does not trim poor quality sequence from the end of your sequencing data. It is always best to check the quality of your sequence in a program that allows you to evaluate the accuracy of the information.
Chromas
Chromas is freely available over the web, and does just about anything you might want to do with .AB1 files. It is an excellent trace viewer for the PC platform (all windows OS's are supported). Follow the link above to download.
EditView from Applied Biosystems Inc.
A free viewer from ABI, this program works on Mac platforms. It allows you to view and edit traces. You can get it directly from Applied Biosystems by clicking on this link.
FinchTV from Geospiza, Inc.
Finch TV is a great trace viewer with versions available for PC or Mac users. Highly recommended. Follow the link above to download.
4Peaks from Neefix Laboratories
This program works only on Mac OS-X, but is useful for viewing and editing chromatograms. It also allows you to print figures from your sequence data. Get the program here.
Configuring your computer to recognize your sequence files:
Windows: download the first chromatogram file, and then double-click it. A window will pop up telling you it doesn't know how to open the file. It will offer to let you browse for the correct application. Do so - choose the chromat viewer you downloaded above. Windows should thenceforth remember what program to use. You can continue to download the rest of your files.
Mac OS X: download the first chromat file, then single-click it and type command-I ('Get Info'). In the subsequent window, click the arrow next to 'Open With:', and in the pop-up menu button underneath, browse for the chromatogram viewer application you downloaded (above). You may wish to click the 'Change All' button, to indicate to your computer that it should always use that application for 'ab1' files.
Mac OS 9 (and earlier): Macs have hidden 'File Type' and 'Creator' codes.
These codes are embedded invisibly in the file structure, and like the Windows 3-character extension, can identify the appropriate program to use for opening a file. Setting these codes can be a bit difficult:
There are many ways to set the File Type/Creator codes:
If you plan to use a web browser (Netscape, Internet Explorer) to download chromatograms, you can configure the browser to set File Type and creator codes:
Netscape: pull 'Edit' to 'Preferences', select 'Applications' (probably under 'Navigator'). Click 'New...' and fill in the blanks with:
- Description: ABI Chromatogram
- Mime Type: application/abi1
- Suffixes: ab1
- Click the 'Application' radio button, then the 'Choose' button. Find your chromatogram viewing program (downloaded as above) and double-click on it. You may also need to select a 'File Type' under 'File Information'. Choose 'ABI1'.
- Close all windows and Netscape should now be able to automatically open your chromatogram-reading program when you download these files.
Internet Explorer: Pull 'Edit' to 'Preferences', click on 'File Helpers' (under 'Receiving Files'). Click 'Add'. Fill in the blanks as follows:
- Description: ABI Chromatogram
- Extension: .ab1
- Mime Type: application/abi1
- Application: click the 'Browse' button, find your chromatogram-viewing program and select it.
- File Type: ABI1 (it may already be filled in correctly from the previous step).
- File Creator: (this is filled in correctly from the 'Application' step).
- Binary Data: click this radio button.
- Use for incoming: click this checkbox.
- Optional: you can also set the Download Destination, if you wish.
- Optional: set 'How to Handle' to either 'Save to Disk' or 'Post Process With Application..'.
- Close all windows and Internet Explorer should now be able to automatically open your chromatogram-reading program when you download *new* files. It doesn't help you with files you may have already downloaded.
If you plan to use an FTP program (or the files are already on your computer), you can use third-party file typers like FileTyper, TypeShuffler, The Associator, DropAttribute or similar. With such a program, set the file type to 'ABI1' and (if you use EditView) set the creator to 'EdVw'.
My sequencing result was not what I expected. How can I tell what went wrong?
The electropherogram is a graphical representation of the fluorescent dye intensity over time. The electropherogram can be used to provide the user with valuable information regarding the quality of their template. Please note, however, when the template does not generate readable results an electropherogram still must be sent for billing purposes. In this case, we truncate the electropherogram to about 30 bases.
Following is a key developed to make analyzing your electropherograms a little simpler.
Noisy Sequence
Your template is "noisy"- the electropherogram will show multiple peaks underneath and on top of each other. This is commonly found with PCR products amplified directly from genomic DNA. If several products are amplified from the template, and a single product is not isolated using either gel extraction or another technique, the primers will sequence multiple products. You can tell that this is the case when you have a normal signal strength, yet there are several peaks in a single location.
- Is it possible that your primer has more than one binding site and you are getting multiple sequences atop one another?
- Is your template contaminated by another fragment that generated peaks underneath the desired sequence?
Not Enough DNA Template
Is your template concentration too low for this reaction, resulting in a low signal-to-noise ratio? (Look at the signal intensity, it should be >100 for each base.)
Your template generated a sequence with a "weak signal strength"- peaks are low and the signal is below 80-100 for each base. Your template is probably too low in concentration for the reaction. The concentrations listed above are for "ideal" templates. Difficult templates often need more DNA just to get through specific regions, e.g. long inserts, secondary structures, homopolymers and microsatellites. An increase in template concentration (10X) can often result in a cleaner sequence, or at least one in which the problem is easier to identify.
- Your primer concentration is probably too low.
- Mismatch in primer sequence. (A single mismatch can cause the signal to decrease by ten-fold)
Homopolymer
Your template generated a sequence with a "homopolymer region". A homopolymer is a region of repeated bases of varying length.
Often, if you get an email mentioning a homopolymer, this means that the enzyme slipped at the homopolymer and the sequence following is noisy and can cause inaccurate base calling by the computer.
A microsatellite region can cause the same problem. There are not too many tricks to get through these regions. The best first step is to run the sample with a primer in the reverse direction so you can obtain sequence on the other side of the region.
Signal Dies Off
Your template generated a sequence that "dies off prematurely"- the sequence will look fine and then either end rapidly before the end of your fragment or slowly towards the end of the fragment.
When the template dies off; there are a few things to think about.
- Is it possible that the enzyme is encountering a secondary structure that it cannot get through?
- Could there be a contaminant left over from the prep of the sample or the sample itself?
G-C Rich Sequence
Is the template G-C rich? There are a few things we can do if this is the case. We can try cycling with a higher temperature to help denature the template. We can run the template with 5% DMSO in order to break the GC bonds. Adding DMSO interferes slightly with the reaction and is not recommended for low concentration templates. Please let us know if you know your template is G-C rich.
Clean Vector, Noisy Insert
 Your template appears to have a "noisy insert with a clean vector"- the electropherogram will be good up until about 70-80 bp and then the template appears noisy.
This problem is usually the product of multiple inserts (PCR of clones from colonies- another colony near by).
When a subcloning procedure allows the insert to be oriented in either direction in the vector, positive colonies can have different sequences. If two positive colonies grow extremely close together and have different orientations of insert, the electropherogram will have clean peaks until the site of insertion where sequences get mixed.
No Sequence
Worst problem of all.your template generated "no readable sequence". This could be caused by a number of different problems.
Some of the more common causes are:
- Not enough template DNA to generate visible results.
- Not enough primer to generate visible results.
- No primer site on the template DNA corresponding to the oligo in the reaction.
- High enough concentration of contaminant to completely cease enzyme activity
I need my DNA template back. Do you still have it?
- The Genomic Analysis and Technology Core will archive DNA templates for 2 months.
- If you can provide the date when the template was run, and the name of the template, we will be able to return it to you (or use it in another sequencing reaction).
- After 2 months we will not be able to access your sample.
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This service is fully automated, using the Beckman Biomek FX liquid handling system. For this reason, it is critical that users of the service adhere to the guidelines below!
Sample submission format:
-DNA to be purified should be brought to the facility in a Nunc V-bottom polypropylene plate (VWR catalog # 62409-108). No other plates may be accepted.
-Sample volume should be brought to 100ml by the user. Failure to do so will result in significant loss of sample during the automated process.
The user may request the purified DNA be returned to them or enter our quantitation and normalization service.
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