Molecular Biology Services


 Molecular Biology Services Overview

 

Molecular biology products and services represent an invaluable tool for the discovery and development of tools and products in the life sciences and medical field. Rockland’s growing suite of molecular biology custom products and services will help accelerate your studies whether working on basic research or performing advanced translational studies. By directly interacting with our molecular biology scientists at every step of the process,  you gain flexibility and reliability during the experimental design and execution of your project. Whether your needs are a simple nucleic acid purification or sequencing project, or assistance with design of a Chimeric Antigen Receptor (CAR) for a  CAR-T project, Rockland’s custom molecular biology services team is ready to help. Our Molecular Biology Services include:

 

 DNA Synthesis button    Gene Cloning Button    Protein Expression button
         
Protein purification button    DNA libraries button    Isolation of Nucleic Acids button
         
 Bioinformatics button    CRISPR button    Custom Services button

 

 DNA Synthesis, Sequencing, Gene Design and Cloning

 

Oligonucleotides, gene fragments, even entire genes and plasmids, can be synthesized according to your custom sequence.  Nucleic acids can also be fully sequenced per your specific requirements.  Synthesized DNA fragments or provided DNA fragments can be cloned into plasmids or other forms of DNA.  

 

  • Oligonucleotide Synthesis - MBS-019
  • Oligonucleotide Sequencing - MBS-020
 

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Gene Cloning 

 
 
Rockland will design, synthesize and clone your gene insert into the desired vector. Alternatively, you can provide your gene insert either as purified DNA, within a purified plasmid or contained in a slab culture and we will ligate it into the appropriate vector.
 
  • Vector Construction - MBS-001
  • Plasmid Isolation using mini-prep system - MBS-002
  • Plasmid Restriction Mapping - MBS-003
 
Gene Cloning

 

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Protein Expression   

 

Native and recombinant proteins can be purified from various sources. Recombinant gene constructions can be generated and produced in the expression system of your choice.

 

 

 E. coli
  • Bacterial protein expression 1L – MBS-004  
 
 Insect Cells
  • Recombinant Baculovirus – MBS-005
  • Plaque assay for viral stock – MBS-006
  • Baculovirus amplification using SF9 – MBS-007
  • Baculovirus MOI Optimization up to 6 small cultures – MBS-008
  • Baculovirus protein expression up to 1L – MBS-009
 
Mammalian Cells
  • Mammalian transient transfection – MBS-010
  • Mammalian stable transfection up to five clones – MBS-011
  • Mammalian retroviral transfection (transduction) – MBS-012
  • Additional Colony expansions per clone - MAB-004
  • Cell expansion and cryopreservation (up to 4 vials) - MAB-084
  • Extended culture time for sample analysis - MAB-019
  • Cell lysate service - MAB-081
  • Cryopreservation of parental cell line (per clone up to 4 vials) - MAB-016
  • Liquid nitrogen storage per clone per year up to 6 vials - MAB-005
  • Master cell banking – MAB-022
  • Mammalian protein expression 1000 sq2 (T-150 x 6) – MBS-013
  • Large Scale Production of Expressed Protein in HeLa cells - MBS-031
  • 7 Liter batch bioreactor - MAB-031
  • Media for bioreactor batch production - MAB-035
  • Time Course and MOI (multiplicity of infection ) Assay - MBS-030
 

 

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 Protein Expression

 

 

 

Protein Purification  

 

Let us do your protein expression or send us the source material and the target protein will be thoroughly purified from contaminating proteins using a multi-step process that includes affinity chromatography.

 

  • Poly His Tag protein purification 1L – MBS-014
  • GST protein purification 1L – MBS-015
  • Custom protein purification – MBS-016
  • Affinity purification – CUST06
  • HPLC purification – CUST12
  • HPLC Analysis – CUST70
  • Concentration by ultrafiltration – CUST80
  • Custom Lyophilization – CUST30
 
Protein Purification


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DNA Libraries 


A cDNA library is generated in one of several available vectors using as little as 20 ng of mRNA.

  •  cDNA Library Construction - MBS-033

 

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Purification and Isolation of Nucleic Acids 


  Genomic DNA or mRNA is isolated from either frozen tissue or tissue culture cell preparations.

   

  • Genomic DNA isolation - MBS-032  
  • mRNA Isolation
  • cDNA Synthesis
 
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Bioinformatics 

 

  • Computational analysis – MBS-018
  • Antigen consultation design of peptide or recombinant protein – CUST95
 
Bioinformatics

 

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Custom CRISPR Gene Editing Services

 

The clustered regularly interspaced short palindromic repeats (CRISPR) is derived from a naturally occurring defense mechanism used by bacteria. The CRISPR/Cas9 system has been adapted to serve as a versatile platform for RNA-directed genome editing in mammalian cells. The Cas9 endonuclease can be programed by a dual RNA (crRNA and tracrRNA), or the core components of these RNAs can also be combined into a single hybrid guide RNA (gRNA). Target recognition is facilitated by the presence of a short sequence called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG. The cleavage of the target DNA by Cas9 triggers two endogenous repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR). The features of these DNA break repair pathways can be exploited to generate gene knock-outs or introduce defined modifications at the site of cleavage.

 

 

 CRISPR Cas9 Targeting System
 
Figure 1. CRISPR-Cas9 targeting system.
In the CRISPR/Cas9 system, a guide RNA hybridizes a 20-nt DNA sequence immediately preceding an NGG DNA motif (protospacer-associated motif or PAM), resulting in a double-strand break (DSB) 3 bp upstream of the NGG. The double-stranded DNA breaks become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR). Adapted from Charpentier & Doudna, Nature, 2013,495:50–1

 

The CRISPR/Cas9 system has a distinct advantage over alternative genome editing technologies such as ZFNs and TALENs. Unlike ZFNs or TALENs, which relies upon the use of customizable DNA-binding protein nucleases, RNA-based CRISPR provide simplicity and adaptability for genome editing. Because of this, CRISPR has rapidly become one of the most popular approaches for genome engineering.

 

Services Offered

 

1. CRISPR design and construction

  • Design 2-5 gRNA to the target of interest with a minimal off-target effects using Optimized CRISPR Design @MIT.  
  • Generate all-in-one construct to drive gRNA and Cas9 expression in one single vector.  To make the stable cell lines, we recommend to use viral or non-viral transposon-based vector for construction.
  • Construct the HDR plasmid for knock-in purpose.
  • Make Cas9 RNP complex (Cas9 protein and gRNA) for quick and more specific gene editing.  

 

2. Generate knock-in and knockout cell lines of interest  

  • Generate knockin and knockout for most of mammalian cell lines: HEK293 cells, CHO cells, HeLa cells or other tumor cell lines etc.
  • Make Gene editing on primary cells: T cells, B cells, NK cells etc. 

Based on your interest, we can choose lipofection, viral transduction or nucleofection to optimize CRISPR delivery. The transfected cells will be selected by antibiotics, MACS or FACS and single clones will be isolated for further validation.


3. Gene-editing validation  

  • On-target confirmation and off-target evaluation. The Surveyor assay will be used to assess the on-target indel frequency. We can also sequence the target regions from selected clones to determine on-target efficacy. In addition, we can further evaluate the target gene editing using Western Blot, ELISA, real-time PCR, or reporter assays etc. 
  • Off-target evaluation. Based on Optimized CRISPR Design algorithm, we choose top 5 off targets. Off-target regions will be PCR-amplified and analyzed by Surveyor assay and sanger sequencing.  Upon request, genomic DNA can also be extracted from targeted clones and processed for targeted deep sequencing. 

 

Service Workflow & Timeline

 

CRISPR Work Flow

 


 

Other Molecular Biology Services 

 

  •  Custom molecular biology services - MBS-017

Do you have a special molecular biology project but can’t find it on our Molecular Biology Services page? Please contact Customer Support for additional details and let us know how we can help.

 

 

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