The Technology

The method of automated DNA Sequencing, utilizes the BigDye Terminator cycle sequencing chemistry from Applied Biosystems (ABI). The samples are sequenced by the 96-capillary 3730xl DNA Analyzer with ABI’s Data collection and Sequence Analysis software.

Free software for viewing the sequencing data – “Sequence Scanner 2” (http://resource.thermofisher.com/page/WE28396_2/)

The following DNA sequencing services are offered:

• Full price Sequencing:

Customers submit samples and primers separately.

Samples in Eppendorf tubes (so we can measure the template concentration) or arranged in strips or plates (mostly for PCR products cleaned by ExoSAP ). Sequencing can be done with in-house DNA sequencing primers or specific sequencing primers provided by the customer.

In this option we will mix DNA, primer, and sequencing reagents, perform the cycling reaction, purify, sequence, analyze, check the outcome and repeat the sequence in case of obvious problem or according to customer request. Customers will receive via email the complete unedited/edited sequence, as will be agreed with each customer.

• Reduced price Sequencing:

Customers submit a mixture of template and primer already “Ready-To-Go” (RTG).

We will mix with the sequencing reagents, perform the cycling reaction, purify, sequence, and analyze the samples. Customers will receive the complete unedited sequence. In this option we will not check the outcome and will not repeat.

• Run-only Sequencing :

Customers submit sequencing plates that are ready to be loaded to on the ABI 3730XL. We will sequence and analyze the samples. Customers will receive the complete unedited sequence via email. In this option we will not check the outcome and will not repeat.

Sample submission

All samples should be submitted with Sample Submission Form for DNA Sequencing.

Write the submitter’s name and the name of DNA/primer on the cap of each tube.

DNA requirements:

Volume: 10µl per reaction

Concentration:

All samples should be dissolved in double distilled water (DDW)

Difficult Templates

If you are having trouble with difficult templates, such as those with high G+C content, homopolymer regions, secondary structures or siRNA and our standard reaction cannot solve the problem, we can run the reaction with 5% DMSO and Betaine. However, we cannot always sequence these regions effectively. Please mark the right column on the submission form.

Primers requirements:

Volume: 10µl per reaction

Concentration: 2 pmoles/µl (2µM)

Primers should be dissolved in double distilled water (DDW).

18-22 bases with 50-60% GC content. Melting point (Tm)= 50-60°C. 

Degenerate primers should be 50 pmoles/µl

Standard Primers available at the DNA sequencing facility:

Instruction for Ready-To-Go mix preparation

RTG-mix should be submitted as 30 ul mixture in tubes/strips/ plates.

In the submission form, RTG-mix should be indicated in the Remarks section.

For more than 8 samples, submission form should be sent both by mail and as printed version.

Please note to list the order of samples in RTG plate by columns (A1 B1 C1 D1 E1 F1 G1 H1 A2 B2 …)

Concentration of template and primers should be adjusted according to the table below.

Results

Sequence data is sent to the customer in both XXX.ab1 and XXX.seq format via E-mail.

Please note the name given by the software to the samples, and the meaning of its parts:

It includes the number of the run, the position of the sample in the sequencing plate, sender’s name, and the templates and primers designations, as filled in the submission form.

For example:

s1287_05_michal-pgem-T7.ab1.seq

Library construction:

We provide full Library construction and sequencing service for NGS, starting at whatever stage required, according to customer’s needs.

When planning a new NGS project we suggest researchers to start with consulting with us. Our team together with a bioinformatics advisor has a reputation of providing expert, personalized service from the first stages of experimental design, choice of library preparation method and sequencing parameters.

Our experienced stuff have years of experience with most of the commercial kits available on the market. Some commercial kits are in standard usage and in stock in our lab and can be ordered in any numbers upon request. Other commercial kits can be ordered by customers and sent to our lab together with the samples for efficient and reliable handling. Service for homemade protocols is also available, if a detailed manual is provided.

Our in-house stock Libraries:

               TruSeq kit of Illumina

                 https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-rna-v2.html

Instruction for samples delivery:

The minimal RNA amount for library generation is 100ng dilute in 52ul.

The maximal RNA amount for library generation is 2ug dilute in 52ul.

Samples should be delivered in strips only marked in chronological numbers.

In the submission form , fill the table with your sample name respectively to the chronological numbers marked on the strip.  

               Sense kit of lexogen

               https://www.lexogen.com/sense-mrna-sequencing/

Instruction for samples delivery:

The minimal RNA amount for library generation is 500ng dilute in 52ul.

The maximal RNA amount for library generation is 2ug dilute in 52ul.

Samples should be delivered in strips only marked in chronological numbers.

In the submission form , fill the table with your sample name respectively to the chronological numbers marked on the strip.  

NEBNext Multiplex Small RNA Library prep kit of New England

https://international.neb.com/products/e7300-nebnext-multiplex-small-rna-library-prep-set-for-illumina-set-1#Protocols%20&%20Manuals

Instruction for samples delivery:

The minimal RNA amount for library generation is 100ng dilute in 7ul.

The maximal RNA amount for library generation is 1ug dilute in 7ul.

Samples should be delivered in strips only marked in chronological numbers.

In the submission formלצרף לינק לטופס הגשת דוגמאות , fill the table with your sample name respectively to the chronological numbers marked on the strip.  

Nextera kit of Illumina

https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-dna.html

Instruction for samples delivery:

Samples should be 1-10ng dilute in 20ul.

Samples should be delivered in strips only, marked in chronological numbers.

In the submission formלצרף לינק לטופס הגשת דוגמאות , fill the table with your sample’s name according to its chronological numbers as marked on the strip.  

The Center bases its work on the following instruments:

• The GeneChip® Hybridization Oven 640 - provides precise temperature and cartridge rotation for continuous mixing.
• The GeneChip® Fluidics Station 450 - for the wash and stain operation of Affymetrix GeneChip® arrays. 
• The GeneChip® Scanner 3000 7G with Autoloader - allows to scan higher-density arrays.

Affymetrix Software in the Center:

Affymetrix® GeneChip® Command Console® Software (AGCC)  summarizes probe cell intensity data (.CEL file generation).

Available applications:

• Copy Number Analysis
• Cytogenetics Analysis
• miRNA Profiling
• Transcriptome Profiling

Complete list of applications and arrays (https://www.thermofisher.com/il/en/home/life-science/microarray-analysis.html)

For details please contact the local Affymetrix Product manager:

Amos Grundwag, Eisenberg Bros. Ltd.,

Tel: 972-3-9777037, Mobile: 972-52-8910960,

E-Mail: amosg@eisenbros.co.il

Sample submission:

 All samples should be submitted with Sample Submission Form for Affymetrix Microarrays.

RNA requirements:

RNA

For Eukaryotic Expression assay: 50-500ng (concentration > 50ng/μl)

For Prokaryotic Expression assay: 10-13μg (concentration >500ng/μl)

For Whole Transcript Sense Target assay: 300ng (concentration > 100ng/μl)

For miRNA assay: 0.2-3μg (concentration > 125ng/μl)

Ratio 260/280=1.8-2.0 

RIN (RNA Integrity Number)>7 is acceptable

The Center bases its work on the following instruments:

• StepOnePlus™ Real-Time PCR Systems (https://www.thermofisher.com/order/catalog/product/4376600)
• QuantStudio 3K
• QuantStudio 12K Flex Real-Time PCR System (https://www.thermofisher.com/il/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/quantstudio-12k-flex-real-time-pcr-system.html)
• Fluidigm BioMark™ HD System (https://www.fluidigm.com/products/biomark-hd-system)

The available applications:

• Real-Time PCR using the SYBR Green I chemistry
• Real-Time PCR using the TaqMAN Probe
• SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe
• High-throughput (96x96) RT PCR on Fluidigm BioMark.

Consumables and Disposables

• ABI PrismTM 96-well Optical Reaction Plates sealed with Optical Adhesive Covers
• ABI PrismTM 384-well Optical Reaction Plates sealed with Optical Adhesive Covers
• Micro Fluidic Cards

Real-Time PCR using the SYBR Green or TaqMAN:

There are three options:

1. Running a 96-wells PCR plate, the assay is done by the customer.
2. RNA samples are submitted. RT-PCR amplifications and Real-Time PCR are performed at the Center, with primers provided by the customer.
3. cDNA samples are submitted. Real-Time PCR is performed at the Center, with primers provided by the customer.

Requirements for Primers (SYBR-GREEN chemistry)

• From 30 to 80% G/C content.
• Melting temperature (Tm) from 58 to 60°C.
• The five nucleotides at the 3´ end of primer should have no more than two G or C bases.
• Recommended primer concentration 50nM.  
• Length of amplicons should be in 50-150 basepairs range.

Requirements for Primers and Probe (TaqMAN chemistry)

• Primers and probe with 30 to 80% G/C content
• Melting temperature (Tm) for the primers 58 to 60°C, Tm for the probe 8 to 10°C higher.
• Do not select probes with G on the 5´ end.
• The five nucleotides at the 3´ end of primer should have no more than two G or C bases.
• Recommended primer concentration 900nM and probe concentration 250nM.
• Amplicons are in the 50- to 150-basepairs range.   

SNP (Single Nucleotide Polymorphism) detection using the TaqMAN Probe

SNP Assays provided by the customer.

Genomic DNA samples are submitted

The DNA concentration should be 10-20 ng/µl.

The volume of the samples depends on the number of requested SNPs but not less than 20 µl.

DNA samples should arrive in a 96-wells plate even if the plate is not full.

The order of the samples in the plate should always be as follows:

          Sample 1 is in position A1

          Sample 2 is in position B1

          .....................................

          Sample 96 is in position H12

High-throughput (96x96) RT PCR on Fluidigm BioMark.

Real-Time PCR is performed at the Center.

IFC-array provided by the customer.

For Gene Expression analysis the customer need to provide plate with cDNA samples and plate with diluted primers. The final concentration of Forward+Reverse primers should be 50µM.

Fragment Analysis

The Center bases its work on the following instrument: 96-capillary 3730xl DNA Analyzer

Software:

• GeneMapper® Software can be purchased through the local agent Rhenium (www.rhenium.co.il) 

• Peak Scanner™ 2 Software can be free downloaded from here: 

Dye sets:

The two dye sets for detection and sizing labeled PCR product are available in the Center:

• DS-30: Size standard ROX-400. PCR product should be <400bp and labeled with 6-FAMTM, HEXTM, NEDTM.

• DS-33: Size standard LIZ-500. PCR product should be <500bp and labeled with 6-FAMTM, VICTM, NEDTM, PETTM.

Sample submission:

All samples should be submitted with Sample submission form for labeled PCR products

1. PCR is performed by customer

Labeled PCR amplification products are submitted.
PCR products should arrive in a 96-wells plate even if the plate is not full.
The order of the samples in the plate should always be as follows:
Sample 1 is in position A1
Sample 2 is in position B1
…
Sample 96 is in position H12

Samples should be diluted appropriately before submission.    
Table with names of the samples should be send by E-mail.

2. PCR is performed at the Center

• DNA samples are submitted. Concentration of DNA >100ng/µl. The samples should arrive in 1.5ml tubes.
• Primers should be provided by the customer. The concentration of the primers should exceed 10µM and be noted on the tube. 

Currently unavailable.

The iPrep™ Purification Instrument (http://tools.thermofisher.com/downloads/B-066973-iPrep_Prfctn_FLR.pdf)

Throughput: 1-12 samples (plus one control) in less than 30 minutes or 192 samples per day.

Reagents: prefilled, sealed reagent cartridges that prevent cross-contamination of samples

Isolation: high quality RNA and DNA from different types of samples

We have two types of kits:

Nano-kit

Quantitative range 25–500 ng/µL

Qualitative range 5–500 ng/µL

Samples per chip-12

Sizing on the 25 – 4000 nt range

Pico-kit

Qualitative range 50–5000 pg/µL in water

Samples per chip-11

Sizing on the 25 – 4000 nt range

All other types of run, for example with Small RNA-kit, can be done with kit purchased by customer.

Sample submission

The RNA samples should arrive only after telephone appointment with the Center.

The samples should be in strips or in RNase free 1.5ml tubes. Keep them frozen all the way.

Sample Volume Required - 2 μL.

If you are planning to work with this sample in future, transfer 2 μL for bioanalyzer to the separate tube.

Always include Sample submission form form Bioanalyzer.