Hi-Di™ Formamide, 25 mL - FAQs

You can make a matrix from running the Sequencing Standard or by running Matrix Standards. To make the Matrix:

(1) After the run, open the DNA Sequencing Analysis Software.

(2) Go to the File menu and select Add Sample(s). Select the file(s) of the standards that you ran, click on the “Add Selected Samples” button, then click the OK box.

(3) After the samples have been added to the Sample Manager, click the Show box, and check the raw data. Peak height should be greater than 200 rfu and the baseline needs to be stable.

(4) Go to the “Tools” menu and select “Make Matrix”. Select the number of files you will be generating the matrix from (e.g., if you are generating it from a sample file, select 1; if from Matrix Standards, select 4).

(5) Click on the “…” box to the right of each line to browse to the sample file(s). Select the file and click “Open”. Do this for all of the samples you will use for the Matrix.

(6) In the “Specify the path for the new matrix file” area, name your matrix. The location the file should be stored in should be the default location and in D:\AppliedBio\Shared\Analysis\Basecaller\Matrix. Note:The matrix file needs to be in both locations in order to autoanalyze the data. If you attempt to point the preferences to look for the matrix in D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller\Matrix, the entire pathway will be written to the .ab1 file for the matrix name and autoanalysis will fail.

(7) Click the “Make Matrix” button. The matrix will either be made correctly or it will generate an error message with specific information on why it failed.

(8) Either copy and paste the Matrix file from the default location to D:\AppliedBio\Shared\Analysis\Basecaller\Matrix or make the matrix 2, the second being saved to that location.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

Clean the capillary window on the capillary using a lint-free tissue (or use Kimwipe®: tissue) to remove all particulates. After cleaning, perform the Test CCD 4-Color test. This is a 5-minute module and should give you 4 lines in the scan window while the module is running. You should expect to see the lines in the following ranges on a scale of 2,000 on the y-axis: Red and Yellow – 700–1,600 and the Green and Blue – 300–1,000 range.

If your results are not close to these ranges, clean the capillary window again. If that fails, then clean the syringe with warm distilled water and change the polymer using a fresh, unexpired bottle. Replace the polymer in the capillary by using the Seq Fill Capillary module and discard the waste water and replace with fresh water.

If all of the above fails, the problem may be hardware related. Please contact Thermo Fisher Scientific Support to initiate a service call.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

Samples can be kept in Hi-Di Formamide at room temperature for up to 48 hours.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

There are two reasons why you might see this message:

Attempting to run Matrix Standards using the P4 RapidSeq (1 mL) E module may result in not having enough peaks being present to generate a successful matrix. If this is the case, try re-running the Matrix Standards using the P4 StdSeq (1 mL) E module or any of the POP-6 modules.

When making the matrix, if you followed the directions for Sequencing Standard but actually ran the Matrix Standards, then you might generate this message since you will have significantly fewer peaks than with Sequencing Standards. Double check the box and tubes that were used and follow the instructions for the standard.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

In order to autoanalyze samples on the 310 Genetic Analyzer, there are three things that need to be done:

(1) Copy the Matrix and Mobility files from the Matrix and Mobility folders located at D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller and paste them into the Matrix and Mobility folders at D:\AppliedBio\Shared\Analysis\Basecaller. If you have not made a matrix yet, you will need to do so prior to running samples.
(2) Open Sequencing Analysis, create an Analysis Protocol, and set it as the Analysis Default.
(3) Set up the Preferences to point to the mobility and matrix files in the local directory and in the General Settings tab, point the Autoanalyze with menu to D:\AppliedBiosystems\SeqA5.4\AppSeqA\Automation310.exe

Once the files are copied and the preferences set, you will be ready to run. At the end of the run, the sample files (.ab1) will be analyzed, but you will not see Sequencing Analysis open. The sample files are analyzed with a non-visible version of Sequencing Analysis.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

A sequencing matrix can be made by either using a Matrix Standard or a Sequencing Standard. The matrix standards consist of 4 tubes, each representing a different nucleotide/dye that have to be run in order to generate a matrix file for analyzing the data.

To make up the Matrix Standard:
(1) Vortex each of the tubes of Matrix Standard for 1 min at full speed.
(2) Add 1 µL of each Matrix Standard into 12 µL of Hi-Di Formamide (so you should end up with 4 tubes, each containing 13 µL).
(3) Heat the mixture at 95 degrees C for 2 min, then cool rapidly by placing on ice.
(4) Run on the instrument using one of the Sequencing Run modules for Filter Set E*.

The Sequencing Standard is DNA that has been sequenced with the chemistry you want to use (e.g., BigDye Terminator v.3.1), cleaned up, and dried down. After reconstitution, you can run it like a regular sample, use the sample file to make the matrix, and then apply the matrix back to the sample file to see how well it works. The Sequencing Standard can also be used as a positive control to run on your instrument.

To make up the Sequencing Standard:
(1) Add 100 µL of Hi-Di Formamide to a tube containing the lyophilized Sequencing Standard.
(2) Vortex at full speed for at least 1 min (the DNA can be difficult to resuspend, mixing for a shorter time may result in weaker signal and a poor quality matrix).
(3) Heat at 95 degrees C for 2 minutes then cool rapidly by placing on ice.
(4) Run 10 µL using the appropriate mobility file and run module for Filter Set E*.
Note: If making a matrix using the Sequencing Standard, do not use the P4 RapidSeq (1 mL) E module.
(5) If the raw signal intensity is >4000 rfu, take 10 µL of the Sequencing Standard and add it to 90 µL of Hi-Di Formamide and re-run the standard.

*The BigDye Terminator v.1.1 and v.3.1 chemistries are spectrally similar, so they can be run under the same Filter Set. However, you will need a different matrix for each chemistry.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

We recommend storing Hi-Di Formamide at -25 to -15 degrees C. Regarding shelf life, this product is covered under our general 1-year warranty and is guaranteed to be fully functional for 12 months from the date of shipment, if stored as recommended. Please see section 8.1 of our Terms & Conditions of Sale for more details.

The shelf life of the aliquoted product is up to 3 months at -25 to -15 degrees C as this would depend upon how the product is handled, i.e., conditions under which the aliquoting is performed, whether the tip and tubes are free of contaminants, etc.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center and Next-Generation Sequencing Support Center.

We recommend using Hi‑Di Formamide due to the sample stability in the solution. 0.1 mM EDTA, pH 8, can also be used as an injection solution but is more susceptible to evaporation. The use of water as an injection solution causes highly variable quantities of DNA to be injected, because there is no competition for the charged DNA/salts.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Once the sample has been resuspended in Hi‑Di Formamide, we recommend that you load it immediately on the instrument. It should be stable for 24 hours on the instrument.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

In GeneMapper Software, “failed sizing” is a result of the software being unable to identify the size standard peaks as defined by the selected size Standard. Please confirm that the size standard used in the sample matches the size standard selected in GeneMapper Software. The Size Match Editor function in GeneMapper Software allows you to view the size standard peaks sizing assignments. In the Size Match Editor window, the size standard peak heights can be reviewed, the user can determine if all the peaks in the size standard are present, or if there are any extraneous peaks in the same color as the size standard that are present. The Size Match Editor can be accessed by highlighting the failed samples in the Samples tab, and then going to the Analysis pull down menu > Size Match Editor.

-Within the GeneMapper Software's Analysis Method, the Peak Detector tab contains the minimum peak height setting for each color (blue, green, yellow, red, purple, and orange). If the size standard peaks are below the minimum peak height setting on the Y-axis, typically 50, the software will ignore these peaks (this threshold is different for fragment analysis that occurs in the 3500 Data Collection software). Although this setting may be altered, it may be necessary to troubleshoot further before doing so. If the sample has strong signal and the size standard is weak, the sample may be preferentially injecting into the capillaries. The sample may have a high salt concentration and/or robust amplification. To confirm, a size standard-only sample should be run to confirm the performance of the size standard alone. If the signal intensity of the size standard improves, the sample may require desalting or less PCR product should be used. If the size standard-only sample still shows low signal, the issue may be possible degradation of the size standard or the Hi-Di Formamide and they may need to be replaced. It may also be a sign that the capillary array is old or clogged.
-If there are any extraneous peaks, this can be due to pull-up peaks from overloaded samples. Extraneous peaks in the same color as the size standard dye label may interfere with the sizing algorithm in the analysis software. The sample can be reduced to prevent the pull-up peak from overloaded samples. If the sample is not overloaded, please confirm that the fluorescent dye in use is part of the dye set selected. The spectral calibration may also need to be re-run.
- If the larger peaks in the size standard are missing, it can also cause the sizing analysis to fail. This could be sample-related where smaller ions and PCR products are preferentially injected into the capillaries and out-compete the injection of the larger products. To confirm, the size standard should be run without the sample.
- If a size standard-only injection is performed, the larger size standard peaks may be missing due to slower migration of the sample. Slower migration can be due to some of the following:
1.Degraded or expired polymer
2.Old array or capillary
3.Old or incorrect concentration of the buffer
4.Old or expired HiDi Formamide
5.Colder ambient temperature
6.Oven temperature too low
If the issue persists, the instrument run time can be increased in order to allow for longer collection time of the largest size standard peaks. However, if the peak resolution is also broader than expected, this may suggest an instrument issue and a service call should be opened.

- If the smaller peaks in the size standard are missing, they may be masked by the primer peak. The easiest fix is to go to the GeneMapper Manager, Size Standard tab, select the size standard definition you are working with and click the “Save As” button at the bottom. Rename the new standard and edit it by deleting any size smaller than 50 bp. Click OK, then Done and change the size standard in your project and re-analyze the data.

The Size Match Editor will allow overriding of the sample quality but it is important to confirm the size standard peaks are correctly identified. Otherwise, the peak sizes of the PCR products may be shifted. To override the sizing quality in the Size Match Editor, click on the “Override SQ” button on the top, middle of the window.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

If the raw data does not show any peaks for sample or size standard, this may have multiple causes:

-A blocked capillary. Run a size standard-only sample to confirm if this is the case.
-A bubble in the capillary may cause delayed migration. A re-injection of the sample can sometimes resolve the lack of data.
-Air bubble at bottom of sample well. Centrifuge the plate before running.
-Autosampler calibration may be necessary.
-Degraded HiDi Formamide. Use properly stored HiDi Formamide.
-High salt concentration. Salt preferentially injects smaller fragments and inhibits injection of larger fragments, so the majority of salt may have been injected in the first injection. Re-inject the sample.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

HiDi Formamide acts as a denaturant and provides sample stability for the heat denaturation step and while on the instrument. DI water is not recommended for injecting fragment analysis samples as it may cause variable injection quality, variable migration of the sample, and is also prone to evaporation.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

For data run on capillary electrophoresis platforms, the troubleshooting process consists of the following steps:

- Identify the problem, i.e., low signal intensity, broad peaks, noisy data etc.
- Check for proper storage of reagents and expiry dates.
- Run the controls. For fragment analysis applications, running the installation standard or size standard on its own can help direct troubleshooting efforts. A possible troubleshooting procedure would be:

1. Mix 12.5 µL of HiDi Formamide and 0.5 µL of Internal Size Standard (i.e., LIZ 600 Dye Size Standard, ROX 500 Dye Size Standard) per well/capillary (for example, A 16 capillary array would need 16 wells, each containing 12.5 µL HiDi Formamide and 0.5 µL of Internal Size Standard).

2. Run the standards using Standard Run Modules. Sizing should pass in the Analysis Software using a Default Analysis Method and Size Standard Definition.

This can be used to determine if the size standards, instrument hardware, and consumables on the instrument (i.e., polymer, buffer, capillary/array) are working properly and if the proper software settings have been selected for the analysis. If the standards do not look good and weekly maintenance has not been performed, perform the weekly maintenance and re-run the standards. If the standards still do not look good, contact Technical Support at techsupport@thermofisher.com. If the installation/size standard plate looks good, go to step 3:

3. Set up a plate of PCR reactions using a laboratory internal DNA control sample or your QC DNA, as per your laboratory's established protocol.

4. After the PCR reaction is complete, dilute the sample as per your laboratory protocol and mix 1 µL of diluted sample, 0.5 µL Internal Size Standard, and 10.5 µL HiDi Formamide. Alternatively, the diluted sample can be added to the size standard-only plate prepared in step 1, to save on reagents.

5. Denature the sample at 95 degrees C for 3 minutes, and place on ice for 3 minutes.

6. Run the samples using Standard Run Modules.

This can help determine if the problem is with the chemistry, thermal cycler, template, or the primers. Running the samples on an agarose gel can sometimes help in further defining areas to troubleshoot. If the standards and internal controls all look good but you experience data quality issues with your samples, the problem is most likely in the template or primers being used for that sample. Please contact Technical Support at techsupport@thermofisher.com for further assistance.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Some of the other causes of an Unstable Electrophoresis current detected error message are:

1.Leak on the system
2. Polymer that:
-Has expired
-Has been left on the instrument for more than the recommended time
-Is a mixture of expired polymer and non-expired polymer
3. Running Buffer that was:
-On the instrument longer than 2 weeks
-Not filled to the fill line or evaporated below the fill line
4. An arcing event that was not cleaned afterwards using the water wash wizard
5. Not performing regular maintenance on the instrument
6. Hardware issues

Inspect the system for leaks. If you do not see any leaks on the system, perform the wash the pump chamber and channels wizard using the conditioning pouch and place fresh, non-expired polymer and fresh Anode and Cathode buffer containers. If the problem persists, a service call may be required.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.

If the instrument is not in use, it is not necessary to replace the buffer every 14 days. However, the buffer level needs to remain at the fill line to prevent the capillary or array from drying out. Top off the buffer volume with water if necessary. Replace the anode and cathode buffer containers prior to starting a new run.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Instruments Support Center.