Viral Detection

Specific viral detection (GMP certified)

Our method for detection of specific viral targets using Real-time PCR is based on TaqMan technology so that each virus is detected using a specific set of primers and probe which enables rapid screening and sensitive detection of low levels contaminants (<25 copies per reaction).
Panel of pathogenic viruses is presented below. Additional custom-made assays are available upon request.

List of specific viral qPCR assays

Virus Target Limit of Detection Sample Requirements
Human Immunodeficiency Virus types 1 and 2 (HIV-1, HIV-2) 10 copies Per testing of 8 DNA viral targets:
1 vial of 1ml of non-cell-based
sample or  1-1×10(7) cells per 1ml
or as pellet
Human Papillomavirus types 16 and 18 (HPV-16, HPV-18) 10 copies
Human T-Lymphotropic Virus DNA types 1 and 2 (HTLV-1, HTLV-2) 10 copies
Simian Immunodeficiency Virus (SIV) 10 copies
Mouse Minute virus (MMV) 25 copies
Bovine Polyomavirus (BPyV) 10 copies
Hepatitis B Virus (HBV) 10 copies
Parvovirus B19 (B19) 10 copies
Epstein-Barr Virus (EBV) 10 copies
Human Cytomegalovirus/Herpes Type 5 (hCMV/hHV5) 10 copies
Human Herpesvirus Type 6 (hHV6) 10 copies
Human Herpesvirus Type 7 (hHV7) 10 copies
Human Herpesvirus Type 8 (hHV8) 10 copies
Herpes Simplex Virus types 1 and 2 (HSV-1, HSV-2) 10 copies
Human/Simian Foamy Virus (HFV/SFV) 10 copies
Hepatitis C (HCV) 25 copies

Per testing of 6 RNA viral targets:
1 vial of 1ml of non-cell-based
sample or 1-1×10(7) cells per 1ml
or as pellet

Hepatitis A (HCV) 25 copies
Hepatitis E (HCV) 25 copies

Detection of retroviral activity (GMP certified)

Detection of retroviral contamination in cell-free samples is achieved by the detection of reverse transcriptase (RT) activity using PCR-Based Reverse Transcriptase assay (PBRT, also known as Amp-RT, PERT). The assay is based on RT activity-dependent conversion of target RNA into cDNA that is then detected by quantitative PCR (qPCR).

For more information: Roni Cohen: project-mbc@hylabs.co.il | 054-6565959

Safety and lot release testing

The service laboratories division at hylabs offers broad safety and lot release testing that can meet any stage of the manufacturing process (e.g. master or working cell banks, harvest and final drug product).

With experience of over two decades, hylabs and its leading scientific and quality assurance experts, are driven to deliver the most excellent service in the fastest turnaround time while fully complying with cGMP and ISO 17025 guidelines.

 

Viral safety tests Microbiological tests Genetic assays Other key assays

In vitro adventitious agents

Retroviral infectivity assays
Retroviruses detection
(PBRT)
Specific virus detection
(Real-Time PCR)
Sterility
Endotoxin (LAL)
Mycoplasma
Sequencing
Gene copy number
Gene expression
Cell identity (BARCODE)
Residual DNA
Residual Protein
Next Generation Sequencing
Biodistribution

For more information:

Roni Cohen: project-mbc@hylabs.co.il | 054-6565959

Yael Hadar: yaelh@hylabs.co.il  | 054-5649741

GENEWIZ- GENOMICS SERVICES

Genewiz

 

AZENTA (Genewiz) is known for reliability and completion of projects with a wide range of complexity levels including repetitive sequences with high or low GC content.

 Our 99.9% delivery rate makes our gene synthesis services the top choice for research institutions worldwide.

We provide different genomics solutions

  • Cloned DNA
    -Standard Gene Synthesis and Cloning (PriorityGene) –Gene assembly and Cloning into pUC-57 (Amp/Kan) plasmid or a Custom plasmid of your choice. 

Up to 10kbp.

-Subcloning

-Oligo Pools with HT Cloning

  • Specialized Cloned DNA
    -CRISPR gRNA Construct Synthesis – gRNA Synthesis according to your target sequence and Cloning into pUC-57 (Amp/Kan) plasmid or a Custom plasmid of your choice. 
    -Homology Directed Repair (HDR) Donor Construct and Custom Vector Synthesis
    -Synthetic Libraries – Looking to create a gRNA library to screen knock-outs/knock-ins of a variety of genes in one experiment? GENEWIZ’s custom synthetic library service can build a custom gRNA library for your high-throughput CRISPR screening needs.
  • Linear DNA

-dsDNA Synthesis – (FragmentGENE) – double-stranded, linear DNA fragments of 100-3,000 bp, provided in a lyophilized form that can be easily re-suspended, cloned, and screened to identify the correct clone for downstream applications.

Fast delivery time, lower cost, and greater flexibility. 

-ssDNA Synthesis – ssDNA synthesis service provides up to 10,000 nt’ of full sequence-verified fragments quickly and affordably

  • DNA Libraries

-Synthetic DNA Libraries (Fragment or Cloned)

-Oligonucleotide Cloned Libraries

  • Mutagenesis
    -SDM
    -Substitution/deletion/insertion
  • Plasmid DNA Preparation
  • Codon Optimization – GENEWIZ’s codon optimization tool can optimize multiple critical parameters to stabilize DNA fragments and improve gene expression efficiency.

For more information about our gene synthesis services, don’t hesitate to get in touch with us at:

MolSupport@Hylabs.co.il

 

Sanger Sequencing

Custom DNA Sequencing:

As a leading provider of genomic services, we offer our customers a full range of sequencing services using high-throughput and custom strategies. Projects are treated with the highest level of quality, integrity, and confidentiality.

Submission of DNA Samples for Sequencing

We are using the new ABI 3730xl DNA Analyzer for DNA sequencing. Please pay attention to the following details when sending samples for sequencing.

What to send:

  1. PCR DNA Templates- There are several methods for purifying PCR products: spin column, ethanol precipitation, or enzymatic purification (SAP/Exo I). If more than one PCR product is present, column purification, ethanol precipitation, or enzymatic purification will not isolate the desired product. Use gel purification to isolate the desired product or re-optimize the PCR to obtain a single product. 
    OR
  2. Premix (Primer and Template mixed together)- We encourage customers to submit DNA samples premixed with their specific primer in ddH2O. In this case, submit 15µl per reaction using the following quantity table: Note that PCR Cleanup using Exosap method is not recommended!

Preparation of a Premix 

Target
Primer

Type of DNA

DNA Length(bp)

Amount in 15 µl

Concentration (ng/ µl)

amount

concentration

Volume

PCR Product

100-499

30ng

2ng/μl

30pmol

10 pmol/µl

3µl

500-1000

60ng

4ng/μl

>1000

100ng

6.7ng/μl

Plasmid

3-6k

300ng

20ng/μl

6-10k

600ng

40ng/μl

10-20k

1000ng

66ng/μl

BAC/Cosmid DNA

>20k

750ng

50 ng/μl

15pmol

10 pmol/µl

1.5 µl

 

 

Example
Size of DNA DNA Concentration Primer concentration H2O up to 15µl Total volume
Plasmid 5kb 100ng/µl 10pmol/µl (10µM)    
Amount in Mix 3µl (300ng/15µl) 3µl (30pmol/15µl) 9µl 15µl

Choose the “premix” option in the “service required” section

Template Quantity:

Quantitate DNA by Spectrophotometer (Nanodrop or equivalent). Pure DNA should give an OD260/280 of between 1.8-2.0 and an OD260/230 of about 1.7-3.3. Low 260/280 indicates protein contamination, high OD260/280 indicates possible RNA or residual organics contamination. Low OD260/230 indicates contamination by organics and/or salts.

Regular reaction: DNA & primers in separate tubes

Recommended DNA concentration to send:

Type od DNA 

Amount

Concentration

PCR Fragments100-500bp

30ng/reaction*

10ng/μl

PCR Fragments500-1000bp

60ng/reaction*

20 ng/μl

PCR Fragments>1000bp

200ng/reaction*

25ng/μl

DS Plasmid DNA (3-10Kb)

600ng/reaction*

100-300 ng/μl

DS Plasmid DNA (10-20Kb)

1.2µg/reaction*

100-500 ng/μl

BAC/Cosmid DNA>20kb

1.2µg/reaction*

100-500 ng/μl

*Note that Forward and Reverse primers on one PCR/plasmid sample is considered as two reactions

Primers

  • Provide primers at concentration of 5 pmol/μl in DDW (quantity of 5μl/reaction)
  • Universal primers can be provided by hylabs (please check our primer list)
  • Your Own Primer – guidelines for optimal prime design:

    Verify  the site-specificity of the primer.

    Design primers only from accurate sequence data.

    Search at regions that best reflect your goals. The primer should be located at least 50 nucleotides away from the target sequence or more than 500 nt away.

    Length should be between 18 and 30 nt, with optimal being 20-25 nt.

    Keep the G-C content in the range of 50-65%.

    The Tm should be between 55 C and 65 C.

    Avoid primers that can hybridize to form dimers

    Avoid palindromes because they can form secondary structures.

    Avoid primers with any secondary hybridization sites on the target DNA

There’s a web-based Tm calculator you might try at Metabion oligo calculator

What not to send

  • Samples with high salt concentration (low 260/230 ratio<1.0)
  • Samples with RNA or proteins
  • Samples with traces of ethanol or phenol

Hints and tips for a successful sequencing reaction:

Host Bacterial Strain variability in template preparation

The host strain used for plasmid preparation also impacts template quality. The following information may help you in choosing a host strain:

  • HB101  and DH5a host strains consistently produce good results;
  • XL1 Blue grows slower than most strains and can lead to decreased DNA yields
  • MV1190 and JM109 host strains show some variability in result quality;
  • JM101  (JM100 series) is not recommended.
  • Avoid Terrific broth and other rich media
  • Avoid host strains TG1 and TG2 which contain high carbohydrate levels
  • Template preparation or purification procedures which involve the use of phenol or chloroform should be avoided if possible. If use of phenol or chloroform cannot be avoided an additional ethanol precipitation is recommended.

Effect of low quantity Template:

Excess template can affect data quality when present in sample loading onto the DNA Analyzer. Excess template inhibits the injection of extension fragments thus affecting signals generated from the instrument. Excess template can behave similarly to proteins and accumulate in the capillary array, which affects data resolution.

Effect of Excess Template:

Excess template can affect data quality when present in sample loading onto the DNA Analyzer. Excess template inhibits the injection of extension fragments thus affecting signals generated from the instrument. Excess template can behave similarly to proteins and accumulate in the capillary array, which affects data resolution.

Template Quality:

The 3730 is a capillary-based DNA analyzer that uses electrokinetics for the injection of samples into the capillaries. Therefore your samples must be SALT-FREE AND PROTEIN-FREE. Salt and protein are preferentially injected over DNA and also plug up the capillaries.

  • Do NOT dilute or re-suspend the DNA in TE. Use ddH20 or 1mM Tris pH 8.5.
  • When using the column-based kit, DNA elution should be conducted with ddH2O or with elution buffer diluted 1:10.
  • When using a column-based kit, insure that all of the EtOH is removed before eluting the DNA.
  • If concentrating the DNA insure that all of the EtOH is evaporated before re-suspending the DNA.
  • When using Qiagenminiprep kits, perform the PB wash regardless of the cell line used.

Effect of Residual Salts:

the 3730 DNA Analyzer is especially susceptible to salt in samples from template preparation. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to a lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with extension fragments during electrokinetic injection and result in weak signals.

Effect of Proteins:

many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array adversely affecting data resolution.

Effect of Residual Detergents:

some methods of phage template preparation use detergents such as Triton X-100. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.

Effect of Residual RNA:

residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.

A frequent reason for failure to get good data

Failure results when there is an insufficient level of fluorescent termination products for the computer software to assign a sequence. Some possible reasons:

  • The 3730xl capillary sequencer provides longer reads but is also more sensitive to residual salt and ethanol in the sample. If you are experiencing variable sequencing results try an ethanol PPT or elute in water instead of the supplied Elution Buffer.
  • Too little DNA template results in reactions with little or no signal and poor or no base calling.
  • Too much DNA produces reactions that terminate prematurely, often with fewer than 250 bases of reliable sequence data.
  • Poor quality template DNA. Template DNA must be free of residual ethanol and salt.
  • Insufficient primer concentration or poor quality.
  • The Tm of the primer is << 50°C.
  • The Tm of the primer is >> 65°C.
  • The template does not contain a sequence complementary to the primer.
  • Primer and/or template were not added to the reaction.
  • Using the same primers for sequencing as were used for PCR.
  • A sample containing plasmids from two or more different colonies was submitted inadvertently. CFU’s from densely plated colonies may contain a mixed population of plasmids. This results in two or more overlapping sequences on the electropherogram.
  • A sample containing two or more PCR products was submitted inadvertently. PCR fragment was not gel-purified or during gel purification, the region of the agarose gel from which a PCR fragment is excised and eluted contains a mixed population of fragments.

When to contact us:

The DNA Sequencing Facility is open 5 days/week between 8:00- 17:00.

Please call us or email us if you need any help from our sequencing analysts.

For questions or suggestions please contact us at: lab1@hylabs.co.il, 08-9366475 ext: 222

Online Ordering procedure:

  1. Login to our system using your user name and password.
  2. Fill in your sample name.
  3. Choose the required service.
  4. Choose the type of DNA.
  5. Fill in the concentration of your sample.
  6. Fill in the size of your DNA.
  7. Fill in the Tm of your primer.
  8. When using Universal primers choose from the list (always compare the sequence).
  9. If using your primer, fill in the name.
  10. When acquiring the Bacterial culturing + Miniprep + Sequencing service, please include the desired growth conditions in the comment box.
  11. When done, press the Add Icon.
  12. If you need to correct any of your data, use the edit icon. Make your corrections and then press Add again.
  13. To submit your order, press ‘send form’. Print the form and write the ID number on each tube.
  14. Several minutes after the submission of your order you will receive confirmation of the order and a message informing you when to expect your results.

How to obtain your sequence results:

All the sequence results can be found in your ‘ORDER STATUS & RESULTS’.

Find your last order and press ‘View’ to retrieve your sequencing files. For every sample sent to the sequencing service, two data files will be received:

To access your .ab1 and .seq files click on the required .ab1 and .seq icons. To download all sequences click ‘Download’. Your browser will prompt you to save the zip file to your computer. The Zip files can be opened with any software designed to open zip files. Please make sure to download and save the files to your computer. The files will be removed from our website after two weeks.

  1. One file ends with .seq (fasta format) and contains the sequence data in text format.
  2. The second file ends with .ab1 and contains the densitometric scan (electropherogram) and can be viewed by using the following software applications:
  1. For PC users – http://www.mbio.ncsu.edu/BioEdit/bioedit.html
  2. Chromas PC users – http://www.technelysium.com.au
  3. For Mac users – http://www.appliedbiosystems.com/support/software/dnaseq/installs.cfm

To access your .ab1 and .seq files click on the required .ab1 and .seq icons. To download all sequences click ‘Download‘. Your browser will prompt you to save the zip file to your computer. The Zip files can be opened with any software designed to open zip files. Please make sure to download and save the files to your computer. The files will be removed from our website after two weeks.
 

We also offer a Complementary Test for Sanger Sequencing:

Next-Generation Sequencing (NGS)

Next Generation Sequencing (NGS)

The Next Generation Sequencing (NGS) lab, ISO9001 accredited, provides complete NGS solutions for any project. Whether it’s your first NGS project or your hundredth, we will help you find the right NGS solutions for your research. From planning the experiment to DNA/RNA extraction, library prep, sequencing, and analysis we are here to help you.

Applications include:

  • Microbiome (16s, ITS, 18s): Microbiome analysis, allows one to identify and quantify (relatively) the microbial community in a given set of samples. This is done by sequencing of amplicon libraries for 16s (bacteria/Archaea), ITS (Fungi) or 18s (Eukaryotes). Services included are DNA extraction, library preparation, sequencing, and bioinformatic analysis of the data. 
  • Metagenomics: Whole genome sequencing of DNA extracted from environmental samples, allows for the identification of the microbial community of a sample as well as the de novo assembly of genomes.  Services included are DNA extraction, library preparation, sequencing, and bioinformatic analysis of the data. 
  • Whole genome sequencing: Sequencing of small genomes (phage, bacteria) and large (human) are done by making libraries from the sample, and then sequencing. Services include: DNA extraction, library preparation, sequencing and Bioinformatic analysis
  • Amplicon sequencing: For amplicon sequencing we utilize a two-step PCR protocol, to amplify your region of interest and add the adaptor and index sequences required for Illumina sequencing. Service includes primer design, first and second step PCR, QC of second PCR, sequencing, and analysis.
  • Small RNA sequencing: Small RNA sequencing includes sequencing of microRNAs (miRNA), piwi-interacting RNA, small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or any other non-protein-coding RNA short in length (<200 nt). Services include: RNA extraction, small RNA library prep, sequencing, and analysis. 
  • RNA sequencing/transcriptome:  RNAseq studies are done to compare gene expression levels between different samples, or to study splicing patterns or post-transcriptional modifications. Services include RNA extraction, poly-A selection, rRNA depletion, directional RNA library prep, sequencing, and analysis. 
  • Plasmid sequencing: Next Generation Sequencing (NGS) can be used to sequence plasmids and look for mutations or other changes. A DNA library is prepared from the plasmid, and the library sequenced with a target of 100x coverage. The data is then mapped to the presumed template sequence of the plasmid, or de novo assembled to generate a plasmid map.
  • Gene copy number and insertion site analysis: NGS may be used to determine the number of insertion sites as well as their exact location, using a modified Guides-seq protocol developed at Hy labs.
  •  CRISPR analysis
    – Amplicon sequencing for analysis of the on-target genetic editing events
    – Guide-Seq for analysis of off-target mutation genetic editing events

For more information contact us:

NGS Lab at NGS@hylabs.co.il 073-3238312

For Order click here

Sanger Sequencing – Read More
Clinical Sanger Sequencing 

We also offer  Complementary tests for Next-Generation Sequencing: Sanger Sequencing, QPCR Test.

 

 

 

Clinical Sanger Sequencing

** Our Clinical Sanger Sequencing do not provide diagnostics

Custom DNA Sequencing

As a leading provider of genomic services we offer our customers a full range of sequencing services using high-throughput and custom strategies. Projects are treated with the highest level of quality, integrity and confidentiality.

Submission of DNA Samples for Sequencing

We are using the new ABI 3730xl DNA Analyzer for DNA sequencing. Please pay attention to the following details when sending samples for sequencing.

What to send:

  1. PCR DNA Templates- There are several methods for purifying PCR products: spin column, ethanol precipitation, or enzymatic purification (SAP/Exo I). If more than one PCR product is present, column purification, ethanol precipitation, or enzymatic purification will not isolate the desired product. Use gel purification to isolate the desired product or re-optimize the PCR to obtain a single product.
  2. Premix (Primer and Template mixed together)- We encourage customers to submit DNA samples premixed with their specific primer in ddH2O. In this case submit 15µl per reaction using the following quantity table: Note that PCR Cleanup using Exosap method is not recommended!
  DNA Primers
Type of DNA Amount Concentration Primer amount Primer concentration
PCR Fragments100-500bp 30ng/15µl 2ng/μl 30pmol/15µl 2 pmol/µl
PCR Fragments 500-1000bp 60ng/15µl 4ng/μl 30pmol/15µl 2 pmol/µl
PCR Fragments>1100bp 100ng/15µl 6.7ng/μl 30pmol/15µl 2 pmol/µl
DS Plasmid DNA (3-6Kb) 300ng/15µl 20ng/μl 30pmol/15µl 2 pmol/µl
DS Plasmid DNA (6-10Kb) 600ng/15µl 40ng/μl 30pmol/15µl 2 pmol/µl
DS Plasmid DNA (10-20Kb) 1000ng/15µl 66ng/μl 30pmol/15µl 2 pmol/µl
BAC/Cosmid DNA>20kb 1500ng/30µl 50 ng/μl 30pmol/30µl 1 pmol/µl
Example
Size of DNA DNA Concentration Primer concentration H2O up to 15µl Total volume
Plasmid 5kb 100ng/µl 10pmol/µl (10µM)    
Amount in Mix 3µl (300ng/15µl) 3µl (30pmol/15µl) 9µl 15µl

Choose the “premix” option in the “service required” section

Template Quantity:

Quantitate DNA by Spectrophotometer (Nanodropor equivalent). Pure DNA should give an OD260/280 of between 1.8-2.0 and an OD260/230 of about 1.7-3.3. Low 260/280 indicates protein contamination, high OD260/280 indicates possible RNA or residual organics contamination. Low OD260/230 indicates contamination by organics and/or salts.

Regular reaction (DNA & primers in separete tubes)

Recommended DNA concentration to send:

Type of DNA Amount Concentration
PCR Fragments100-500bp 30ng/reaction* 10ng/μl
PCR Fragments100-500bp 30ng/reaction* 10ng/μl
PCR Fragments100-500bp 30ng/reaction* 10ng/μl
PCR Fragments100-500bp 30ng/reaction* 10ng/μl
PCR Fragments100-500bp 30ng/reaction* 10ng/μl

*Note that Forward and Reverse primers on one PCR/plasmid sample is considered as two reactions

Primers

  • Provide primers at concentration of 5 pmol/μl in DDW (quantity of 5μl/reaction)
  • Universal primers can be provided by hylabs (please check our primer list)
  • Your Own Primer – guidelines for optimal prime design:
  • Verify the site-specificity of the primer.
  • Design primers only from accurate sequence data.
  • Search at regions that best reflect your goals. The primer should be located the primer should be located 50-500 nucleotides from the target sequence.
  • Length should be between 18 and 30 nt, with optimal being 20-25 nt.
  • Keep the G-C content in the range of 50-65%.
  • The Tm should be between 55oC and 65o C.
  • Avoid primers that can hybridize to form dimers.
  • Avoid palindromes because they can form secondary structures.
  • Avoid primers with any secondary hybridization sites on the target DNA

There’s a web-based Tm calculator you might try at Metabion oligo calculator

What not to send

  • Samples with high salt concentration (low 260/230 ratio<1.0)
  • Samples with RNA or proteins
  • Samples with traces of ethanol or phenol

Hints and tips for a successful sequencing reaction:

Host Bacterial Strain variability in template preparation

The host strain used for plasmid preparation also impacts template quality. The following information may help you in choosing a host strain:

  • HB101 and DH5a host strains consistently produce good results;
  • XL1 Blue grows slower than most strains and can lead to decreased DNA yields
  • MV1190 and JM109 host strains show some variability in result quality;
  • JM101 (JM100 series) is not recommended.
  • Avoid Terrific broth and other rich media
  • Avoid host strains TG1 and TG2 which contain high carbohydrate levels
  • Template preparation or purification procedures which involve the use of phenol or chloroform should be avoided if possible. If use of phenol or chloroform cannot be avoided an additional ethanol precipitation is recommended.

Effect of low quantity Template:

Too low quantity template or primer reduces the signal strength and peak height. In the worst case, the signal-to-noise level decreases so that bases cannot be called.

Effect of Excess Template:

Excess template can affect data quality when present in sample loading onto the DNA Analyzer. Excess template inhibits the injection of extension fragments thus affecting signals generated from the instrument. Excess template can behave similarly to proteins and accumulate in the capillary array, which affects data resolution.

Template Quality:

The 3730 is a capillary based DNA analyzer that uses electrokinetics for injection of samples into the capillaries. Therefore, your samples must be SALT FREE AND PROTEIN FREE. Salt and protein are preferentially injected over DNA and also plug up the capillaries.

  • Do NOT dilute or re-suspend the DNA in TE. Use ddH20 or 1mM Tris pH 8.5.
  • When using column-based kit, DNA elution should be conducted with ddH2O or with elution buffer diluted 1:10.
  • When using a column-based kit, ensure that all of the EtOH is removed before eluting the DNA.
  • If concentrating the DNA ensure that all of the EtOH is evaporated before re-suspending the DNA.
  • When using Qiagen miniprep kits, perform the PB wash regardless of the cell line used.

Effect of Residual Salts:

the 3730 DNA Analyzer is especially susceptible to salt in samples from template preparation. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with extension fragments during electrokinetic injection and result in weak signals.

Effect of Proteins

many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array adversely affecting data resolution.

Effect of Residual Detergents

some methods of phage template preparation use detergents such as Triton X-100. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.

Effect of Residual RNA:

residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.

A frequent reason for failure to get good data

Failure results when there is an insufficient level of fluorescent termination products for the computer software to assign a sequence. Some possible reasons:

  • The 3730xl capillary sequencer provides longer reads but is also more sensitive to residual salt and ethanol in sample. If you are experiencing variable sequencing results try an ethanol PPT or elute in water instead of the supplied Elution Buffer.
  • Too little DNA template results in reactions with little or no signal and poor or no base calling.
  • Too much DNA produces reactions that terminate prematurely, often with fewer than 250 bases of reliable sequence data.
  • Poor quality template DNA. Template DNA must be free of residual ethanol and salt.
  • Insufficient primer concentration or poor quality.
  • The Tm of the primer is << 50°C.
  • The Tm of the primer is >> 65°C.
  • The template does not contain a sequence complementary to the primer.
  • Primer and/or template were not added to the reaction.
  • Using the same primers for sequencing as were used for PCR.
  • Sample containing plasmids from two or more different colonies was submitted inadvertently. CFU’s from densely plated colonies may contain a mixed population of plasmids. This results in two or more overlapping sequences on the electropherogram.
  • Sample containing two or more PCR products was submitted inadvertently. PCR fragment was not gel purified or during gel purification the region of the agarose gel from which a PCR fragment is excised and eluted contains a mixed population of fragments.

When to contact us:

The DNA Sequencing Facility is open 5 days/week between 8:00- 17:00.

Please call us or email if you need any help from our sequencing analysts.

For questions or suggestions please contact us at: lab1@hylabs.co.il, 08-9366475 ext: 222

Online Ordering procedure:

  1. Login to our system using your user name and password.
  2. Fill in your sample name.
  3. Choose the required service.
  4. Choose the type of DNA.
  5. Fill in the concentration of your sample.
  6. Fill in the size of your DNA.
  7. Fill in the Tm of your primer.
  8. When using Universal primers choose from the list (always compare the sequence).
  9. If using your primer, fill in the name.
  10. When acquiring the Bacterial culturing + Miniprep + Sequencing service, please include the desired growth conditions in the comment box.
  11. When done, press the Add Icon.
  12. If you need to correct any of your data, use the edit icon. Make your corrections and then press Add again.
  13. To submit your order, press ‘send form’. Print the form and write the ID number on each tube.
  14. Several minutes after the submission of your order you will receive confirmation of the order and a message informing you when to expect your results.

How to obtain your sequence results:

How to obtain your sequence results:

Find your last order and press ‘View’ to retrieve your sequencing files. For every sample sent to the sequencing service, two data files will be received:

  1. One file ends with .seq (fasta format) and contains the sequence data in text format.
  2. The second file ends with .ab1 and contains the densitometric scan (electropherogram) and can be viewed by using the following software applications:
  1. For PC users- http://www.mbio.ncsu.edu/BioEdit/bioedit.html
  2. Chromas PC users- http://www.technelysium.com.au
  3. For Mac users- http://www.appliedbiosystems.com/support/software/dnaseq/installs.cfm

To access your .ab1 and .seq files click on the required .ab1 and .seq icons. To download all sequences click ‘Download’. Your browser will prompt you to save the zip file to your computer. The Zip files can be opened with any software designed to open zip files. Please make sure to download and save the files to your computer. The files will be removed from our website after two weeks.

We also offer a Complementary test for Clinical Sanger Sequencing:

Next-Generation Sequencing

Cell culture

Hylabs provides GMP assays and custom cell culture services to our customers upon request.
For custom experiments, we may assist in planning the requested experiments in order to meet your goals. Among the services offered are:

Variable cell viability assays

Gene editing using, siRNA and CRISPR platforms to provide you with transient or stable cell lines,
Cell signaling, Transfection & infection
Potency testing for pharmaceuticals (POC).
Our tissue culture experiments include the comprehensive output of expression either by real-time-PCR, NGS, Western blots, or ELISA.

Cell authentication analysis.

We also offer Complementary tests for Cell Culture: Virology, QPCR testing, Mycoplasma PCR.

 

 

TOC (Total Organic Carbon)

Total organic carbon (TOC) is the amount of carbon found in an organic compound and is often used as a non-specific indicator of water quality or cleanliness of pharmaceutical and manufacturing equipment.

Hy Laboratories is equipped with the new OI Analytica’s Aurora 1030W TOC Analyzer  for the detection of total organic carbon (TOC) to provide you with the right solution for your TOC need. The system is fully automatic and using heated persulfate oxidation technology to detect TOC in a 0.5-30,000ppm range. The Aurora supports USEPA approved methods: USP  <643> methods  EU 22.44, 23 and complies with the GMP regulation. Hy Laboratories labs are  certified GMP, ISRAC (ISO17025), ISO13485and ISO9001

In  what areas TOC tests are routinely required?

Municipal Waste Water

Monitoring organic carbon of influent facilitates process controls for maximizing plant efficiency, while monitoring effluent is often a requirement for discharging into surface waters.

Industrial Waste Water

Industries which discharge liquid waste into a surface water body are required to monitor TOC.

Power Plants

Limiting potential sources of corrosive compounds can prevent costly damage to expensive equipment.

Pharmaceutical Manufacturers

Water is the most commonly used ingredient in drug production. Regulations limit the concentration of organic carbon to prevent harmful microorganisms from growing.

Electronics Manufacturers

Ultra-pure water is used in the manufacture of microprocessors and computer chips. As processors and circuits become smaller and smaller the water must be kept highly clean to prevent microscopic damage to these miniature circuits.

 Cleaning validation

In the pharmaceutical industry, GMP production requires that the cleaning of drug manufacturing equipment/Medical device will be validated. Many different validation techniques can demonstrate that the manufacturing equipment is cleaned and essentially free from residual active drug substances and all cleaning agents.

TOC analysis can be adapted to any drug compound or cleaning agent that contains carbon. The method is sensitive to the ppb range and is less time consuming than HPLC or UV/Vis. USP TOC methods are standard for Water for Injection and Purified Water, and simple modifications of these methods can be used for cleaning validation.

Water tests

Water is widely used as raw material, inactive ingredient, active pharmaceutical ingredients (APIs) as well as in cleaning applications. The testing of water samples from a water system is critical to the ongoing control of the system and assessment of the quality of the water being used. Control of the microbiological quality of water is important for many of its uses.

At Hy laboratories, we offer testing of the water system which includes bacteriological tests and endotoxin tests. The bacteriological test is performed using mostly membrane filtration method, after that the filter is placed on a suitable agar medium for detection of the total count, coliforms, Pseudomonas aeruginosa, Enterococcus, etc.

Some grades of pharmaceutical waters, such as those used in parenteral applications (e.g., Water for Injection, Water for Hemodialysis, and the sterilized packaged waters made from Water for Injection) strictly limit the number of endotoxins. BET (bacterial endotoxin test) is done using any of the methods suitable for water: Gel clot, turbidimetric or chromogenic.

Hy Laboratories also offer the service of our skilled and certified samplers. Our samplers will come to your facility and perform sampling of the water system, then delivers it to the lab, in a timely manner, to perform the analysis.

We also offer Complementary tests for Water Tests: Bacterial Identification, Ecology, and Morphology.

 

Virology

virology

 

Our virology laboratory is ISO 9001 and ISO 17025 accredited, cGMP-compliant and operates at a BSL-2+ facility.

We perform tests and design unique projects, based on ASTM and ISO standards, for our variety of customers ranging from pharma, cell therapy and startup companies to basic and academic researchers.

Antiviral Testing – for the assessment of the antiviral potential of products and devices, such as air filtration and purifiers, disinfection devices, sterilizers, liquid preparations and formulas, disinfectants, porous and non-porous surfaces, textiles and face masks.

In-Vitro Adventitious Virus Testing Assays – as part of our safety and lot release portfolio of tests, we provide a 14-days adventitious agents assay, by challenging human and primate cell cultures with any biological product, for the detection of possible viral contaminants present in low titers.

Viral Clearance and Viral Disinfection Assays – testing the capacity of the production or of the disinfection process to remove or inactivate viruses from devices and products, as well as testing at key stages of production for freedom from detectable infective viruses.

Lot Production and Titration – we cultivate, produce and quantify titer for a variety of infective viruses, at small to large scale production.

Retroviral Infectivity Assays – complementary test for our reverse transcriptase detection assay (PBRT) for product and lot release.

Our services employ valid quantitative methods for measuring viral viability and infectivity upon various treatments and conditions, such as titer determination in TCID50/ml unit or PFU/ml unit (plaque assay) and supportive molecular verification methods for viral nucleic acid detection (for example Real-Time PCR).

Our virology laboratory utilizes strains of human coronavirus, human influenza, avian and bovine viruses, and retroviruses. We provide services for any viral portfolio required per customer request.

 

We also offer Complementary tests for Virology: Cell Culture, QPCR Testing