Vector Cloning

세계 최대의 맞춤형 벡터 클로닝 서비스 회사인 VectorBuilder는 연구 요구사항에 맞는 거의 모든 맞춤형 DNA 벡터를 실제로 클로닝할 수 있다. 당사 플랫폼의 약 200개의 벡터 시스템은 광범위한 R&D 작업을 통해 최적화되었으며, 모든 벡터는 in vitro 및 해당되는 경우 in vivo 테스트를 통해 검증되었다. 경제적인 가격과 빠른 처리시간을 갖는 VectorBuilder에 클로닝을 아웃소싱하면 시약 및 인건비 측면에서 실험실의 DIY 클로닝보다 저렴할 뿐 아니라 클로닝과 관련된 시간과 노력 등을 줄여준다.DNA 벡터의 클로닝 외에도 plasmid DNA preparation, virus packaging (lentivirus, AAV, adenovirus, MMLV, baculovirus, etc.), library construction, BAC recombineering, mutagenesis, stable cell line generation 및 RNA preparation과 같은 많은 관련 서비스를 제공한다.

Service Highlights

  • 다양한 어플리케이션을 위한 맞춤형 벡터를 빠르게 디자인할 수 있는 매우 직관적인 무료 온라인 Vector Design Studio 이다.
  • 방대한 vector backbones 및 vector components를 통해 클로닝에 소요되는 시간과 비용을 절약할 수 있다 (프로젝트의 약 20% 만이 de novo 유전자 합성을 필요로 함).
  • 신뢰할 수 있는 실험 결과를 제공하는 기능적으로 검증된 벡터 백본 및 컴포넌트.
  • 단순한 디자인부터 복잡한 디자인까지 벡터를 생성하기 위한 다양한 클로닝 기술 활용.
  • 까다로운 벡터 클로닝에 대한 광범위한 경험과 노하우.
  • Plasmid DNA preparation 및 virus packaging과 같은 downstream 서비스를 쉽게 제공받을 수 있다.
  • 100% sequence guarantee.
  • 최대 6개월 동안 무료로 벡터 보관.
  • Custom vector의 IP는 고객 소유.

Service Details

Price and turnaround

Custom 벡터 클로닝의 워크플로우는 일반적으로 고객이 제공한 시료의 QC (해당되는 경우), 필요한 벡터 컴포넌트의 구매 (해당되는 경우), 실제 벡터 클로닝의 세 부분으로 구성된다. 가격 및 소요시간에 대한 간략한 개요는 아래 Table 1에 나와 있다.

Table 1. Overview of price and turnaround for custom cloning.

Vector cloning* Vector component sourcing (if applicable) QC of customer-supplied materials (if applicable)
Type Depends on the complexity of vector design  In-stock template Customer-supplied template De novo gene synthesis As vector backbone As source for vector component
Price

a. For shRNA, gRNA and short fragment expression (e.g. enhancer, promoter): starting from $99

b. For protein expression: starting from $159

$0-$50 for >80% of templates $0 Refer to Table 3 $150 Starting from $120
Turnaround** 1-3 weeks for >80% of the projects Immediately available Immediately available after passing QC Refer to Table 3 3-5 days
Description Cloning methods include PCR, ligation-based cloning, Gibson, Gateway and Golden Gate cloning In-stock templates include promoters, ORFs, markers, linkers and protein tags. About 20% of projects need de novo gene synthesis QC includes quantification, re-transformation, plasmid prep, RE digestion and Sanger sequencing.

Note:

* VectorBuilder의 표준 백본 및 컴포넌트로 제작된 벡터는 아래 Table 2를 기준으로 동일한 가격과 처리시간이 적용된다.

** 클로닝 처리시간은 생산 시작부터 완료까지의 시간을 의미한다. 고객이 제공한 시료의 운송 시간과 QC, 최종 결과물을 고객에게 배송하기 위한 운송 시간은 포함되지 않다.

Table 2. Price and turnaround for simple vector cloning.

Vector Type Price Turnaround
shRNA vector $99 7-12 days
gRNA vector (single-gRNA) $99 6-11 days
gRNA vector (dual-gRNA) $299 10-17 days
Expression vector $159 7-13 days

Table 3. Price and turnaround for de novo gene synthesis.

Fragment length Price (USD)* Turnaround
<1.5 kb $0.12/bp 6-10 days
1.5-3 kb $0.14/bp 10-14 days
3-5 kb $0.16/bp 10-16 days
5-7 kb $0.18/bp 16-20 days

*The de novo gene synthesis fee may be higher when 1) the fragment contains regions that are difficult to synthesize such as high GC content, simple repeats or segmental repeats; 2) fewer than three DNA fragments are synthesized.

Deliverables

기본 제공 형식은 Plasmid DNA solution이다.  Miniprep (> 10ug), Midiprep (> 100ug), Maxiprep (> 300ug), Megaprep (> 1mg) 및 Gigaprep (> 10mg)의 여러 스케일의 high-quality plasmid DNA 를 추가로 구입할 수 있다. 바이러스 벡터의 경우 다양한 스케일로 제공되는 바이러스 패키징 virus packaging 이 downstream 서비스로 제공된다.

Quality control

VectorBuilder에서 클로닝한 모든 벡터는 100% 서열을 보장한다. 당사의 벡터는 디자인한 대로 정확한 서열을 포함하도록 엄격한 품질 관리를 받는니다. 일반적인 QC에는 DNA 정량, A260/280 측정, 제한효소 처리하지 않은 또는 제한효소로 처리한 plasmid DNA의 전기영동, Sanger 시퀀싱, glycerol 스톡에서 박테리아 회수 및 re-transformation이 포함된다.

Customers-supplied materials

백본이나 유전자 템플릿 등 고객이 제공한 시료가 필요한 경우 "서포트" > "시료 제출 양식". 에서 시료의 정보를 제공해 주세요. 시료의 지연이나 손상을 방지하기 위해 가이드라인을 엄격히 따라 주시기 바랍니다. 모든 고객 제공 시료는 VectorBuilder에 의해 필수 QC를 거치며, 아이템 유형 및 용도에 따라 항목당 $ 120부터 시작하는 추가 QC 요금이 발생할 수 있다. 고객이 제공한 시료가 QC를 통과할 때까지는 생산을 시작할 수 없다.

FAQ

What methods does VectorBuilder use for its vector cloning services?

At VectorBuilder, a combination of various molecular biology techniques is utilized to generate custom vectors based on customer needs. While expression vectors are typically constructed using Gateway cloning or Gibson assembly, shRNA and gRNA vectors are cloned using a ligation-based approach to provide you with your desired vectors at the lowest prices and fastest turnaround times utilizing our highly optimized high-throughput cloning platform. Additionally, we routinely use a variety of other methods including de novo gene synthesis and Golden Gate cloning as needed, on a project-by-project basis.

Which bacterial strain does VectorBuilder use for cloning its vectors? What bacterial strains can I use for propagating my plasmid obtained from VectorBuilder?

All vectors at VectorBuilder are cloned using our proprietary host strain VB UltraStable, which is designed to achieve high transformation efficiency (>1x109 cfu/ug) and for propagating DNA plasmids with unstable elements such as repeated sequences. Therefore, we highly recommend that you use VB UltraStable chemically competent cells for propagating your plasmids obtained from VectorBuilder. However, our vectors are compatible with and can be propagated in other commonly used bacterial hosts strains such as DH5α and Stbl3.

Why is the yield of my plasmid prep so low?
The origin of replication on your plasmid is for low-copy replication

The number of plasmid copies per bacterial cell is determined by the origin of replication on the plasmid. Some origins have inherent low copy number. Check the copy number of the origin of replication on your plasmid. For low-copy plasmids, increase the amount of E. coli culture for plasmid DNA prep in order to obtain satisfying DNA yield.

Check the copy number of the origin of replication of plasmids supplied by VectorBuilder

The volume of bacterial culture is too low for plasmid prep column

Please check the binding capacity of your plasmid prep column and whether your plasmid is high- or low-copy plasmid. For mini preps, we recommend that you harvest 1-5 ml of overnight bacterial culture. For maxi preps, if the plasmid is high-copy plasmid, we recommend using 100-150 ml of overnight bacterial culture; if the plasmid is low-copy plasmid, we recommend using 300-500 ml of overnight bacterial culture. Typically, for high-copy plasmid, ~5 ug of plasmid DNA can be extracted from every 1 ml of culture in mini prep and ~500 ug of plasmid DNA can be obtained from 150 ml of culture; for low-copy plasmid (e.g. pET), 1.5-2.5 ug of plasmid DNA can be harvested from every 1 ml of culture in mini prep and 150-200 ug of plasmid DNA can be obtained from 150 ml of culture.

Only a fraction of bacteria in the liquid culture contain plasmids

Some antibiotics, ampicillin in particular, degrade fast in liquid culture. As a result, bacteria that do not contain plasmids can propagate to a significant fraction of the culture, causing poor yield of the plasmid prep. To avoid this, please prepare ampicillin containing growth medium freshly before use and make sure that enough ampicillin is supplied. Also, when culturing ampicillin-resistant bacteria, do not let the liquid culture saturate for too long before harvesting. Besides insufficient antibiotics in the culture, extracting plasmid DNA from very old culture can also result in low yield, since many bacterial cells are dead and plasmid DNA they contain is degraded. Therefore, try to extract plasmid DNA from fresh culture. If plasmid prep cannot be performed immediately, you can spin down the bacteria and store the pellet in -80°C freezer for later plasmid prep.

The liquid culture is directly inoculated from E. coli stab culture

Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. Detailed user instructions can be found by going to menu item Learning Center > Documentation > Stab Culture.

You have not carefully followed the manual of the plasmid prep kit

If you use a plasmid prep kit, please carefully read the manual before use. Improper operations can often lead to poor performance of the kit.

The mini/maxi prep column is low-quality

Some brands of plasmid DNA prep columns perform poorly or inconsistently for DNA preparation.