Molecular Biology Services

1.Gene Cloning

Sangon Biotech have developed particular technology for obtain DNA fragments ("gene") with specific functions either by cloning from natural existing organism or by chemical gene synthesis. Cloning is usually more convenient than gene synthesis, but cloning is also time-consuming and sometimes can be difficult. Sangon Biotech not only help you to obtain the desired gene, but also help you to subcloning the desired gene into any vectors to save your time and money.

• Direct gene cloning with known sequence from known source
• Norvel gene cloning using RACE (Rapid Amplification of cDNA Ends) technology

2.Gene Expression Analysis Using Real-time Fluorescent Quantitative RT-PCR

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product (either proteins or non-protein coding genes such as miRNA). The regulation of gene expression process was strictly controlled in a complicated network in vivo. Gene expression profiling indicates the role of a particular gene and the changes in gene expression is associated with many biological process such as disease developing. So monitor the changes in gene expression becoming an more and more important method to monitor the "action" of particular genes.

Real-time PCR analysis is the gold standard for quantifying gene expression. We have developed methods for sensitive, accurate quantification of mRNA or microRNA using real-time PCR. Both TaqMan probe-based analysis and SYBR Green dye-based analysis are available. Individual samples or 96 or 384 well plates can be analyzed.

• mRNA Real Time Fluorescent Quantitative RT-PCR
• microRNA Real Time Fluorescent Quantitative RT-PCR

3.Genotyping

Microsatellite (STR) Genotyping

Microsatellites, also known as short tandem repeats (STR), are widely used molecular markers in genetics. Sangon Biotech offers a complete microsatellite (MS) genotyping service.

Single Nucleotide Polymorphism (SNP) Genotying

SNPs are one of the most common types of genetic variation which is associated with many diseases. SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. Sangon Biotech have developed several SNP genotyping platforms for different requirement including:

• Direct Sequencing
• Restricted Fragment Length Polymorphisms (RFLP)
• TaqMan Probe
• Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

4.cDNA Library Construction & EST Sequencing

cDNA library construction is a powful tool for gene expression, gene function research, norvel gene characterization, and many other applications. But obtain high-quality cDNA libraries is time-consuming and can be very challenging. Sangon Biotech has over 10 years of experience in generating cDNA libraries from various of samples. Thanks to our comprehensive sequencing platform. cDNA libraries with ESTs (expressed sequence tag) can also be sequenced and analysized here.

5.Species identification & Microbial Population Analyzing

• Identification of Bacteria or Fungi
• PCR-DGGE (Denaturing Gradient Gel Electrophoresis) analysis for the identification of microbial populations of Bacteria
• Microbial Population Analyzing

6.Suppression Subtractive hybridization(SSH) Service

7.Single Nucleotide Polymorphism(SNP) genotying Service

SNP service content price and procedure

SNP testing service platform

• Direct sequencing
• Restricted fragment length polymorphisms (RLFP)
• TaqMan Probe
• MALDI-TOF MS

8.Illumina beadchip Services

• Illumina beadchip SNP genotyping
• Illumina beadchip methylation
• Illumina beadchip gene expression profiles
• Illumina beadchip microRNA expression profiles

9.Methylation Analysis

• Bisulfite Sequencing Method (BSP)
• Methylation Specific PCR Method (MSP)

10.Virus packaging

• Adenovirus packaging services
• Lentiviral packaging services

11.RNA Interference (siRNA) Experimental Services

1.Gene Cloning

Services	Price ($)	Turnaround Time	Remarks
Known gene cloning from Prokaryotic or eukaryotic genome	Minimum charge at $80 (<=1,000 bp) For gene length >1,000 bp, $0.1 / extra bp	15 business days	Extra charge for high GC
Known gene cloning from Eukaryotes cDNA	Minimum charge at $130 (<=500 bp) For gene length >500, $0.17 / extra bp	20 business days	Extra charge for high GC
5'RACE	Minimum charge at $800 (<=500 bp) For fragments >500 bp, $0.34 / extra bp	1 to 2 months	Including 3-5 designs of specific primers, RACE PCR and cloning sequencing.
3'RACE	Minimum charge at $500 (<=800 bp) For fragments >800 bp, $0.17 / extra bp	1 to 2 months

All data (including gel analysis photos of PCR, Sequencing trace files, etc) will be provided.

2.Gene Expression Analysis

Project		Quantity	Price ($)
Primer charge	mRNA	Pair	$8.5/pair
	miRNA	Pair	$13.5/pair
	Probe	Probe	$165/probe
DNA extraction	Blood	sample	$3.35/sample
	Cells, animal tissues, etc.	sample	$5/sample
	Water, mud, soil samples	sample	$5~$8.35/sample
RNA extraction + reverse transcriptase		sample	$13.5/sample
plasmid construction (required by Absolute quantification method)		plasmid	$16.5/plasmid
Standard Curve**	2 repeats	#gene* 5 point*2 repeat	$5/reaction
	3 repeats	#gene* 5 point*3 repeat	$5/reaction
qPCR of Samples	1 repeats	#Sample * #gene *1 repeat	$5/reaction
	2 repeats	#Sample * #gene* 2 repeat	$5/reaction
	3 repeats	#Sample * #gene* 3 repeat	$5/reaction

**By default, 5 gradient dilution is used.

3.Microsatellite (STR) Genotyping

Steps	Content	Price ($)
DNA extraction	Whole blood samples	$3.5 /sample
	Paraffin sections, tissue samples	$8.5 /sample
Pre-test	optimize PCR reaction conditions	$33.5 for each amplified region
PCR reaction	According to preliminary results of a large number of PCR	Tube / $0.85 ~ $1.7 (based on the difficulty of PCR)
Label primers	Synthesis of fluorescent labeled primers	$50~$83.5/oligo
Formal test	STR genotyping using ABI 3730XL DNA analyzer	$250 /96 reaction (without internal standard), minimum charge: 96 reaction
Requirements for samples	Genomic DNA	Concentration > 20 ng/μl, amount > 20 μl (Specific amounts required according to experimental requirements)
	Whole blood samples	1 ml anticoagulated blood (please use EDTA as anticoagulant)
	Paraffin sections, tissue samples	>100 mg
Experimental period	4-6 weeks (Specific time accord to STR loci /sample number)

All data including excel table with site statistics situation, sequencing trace files and original documents will be provided.

4.Library Construction

Project	Content	Price ($)
SMART cDNA library	cDNA library construction	$2500 (provide 20 sequencing reactions for free)
Normalized cDNA library	Normalized cDNA library construction	$13,000 (provide 20 sequencing reactions for free)
EST sequencing	<5000 clones	$3.2 per clone
	5000-10000 clones	$3.7 per clone
	>10000 clones	Inquire
	Standard bioinformatics analysis	Free
	Advanced bioinformatics analysis	Inquire
Sample Requirement	High quality samples are required, Tissue: >1 g Cultured cells: >1 * 107 total RNA: > 20μg
Experimental period	4-6 weeks

5.Strain indenification & Microbial Population analyzing Services

5.1Strain identification

a.Services

• Full-length 16S rDNA sequencing: The full-length 16S rDNA (~ 1,500 bp) was amplified and cloned into cloning vector by TA-cloning and followed by Sanger sequencing. More comprehensive information could be obtained by analyzing full-length 16S rDNA sequence.
• Fungal ITS analysis: Identification of fungal by sequence of ITS (Internal transcribed spacer) region..
• Fungal 18S rDNA analysis: Similar with 16S rDNA analysis of bacteria, 18S rDNA of Fungal is widely used for species identification.
• Fungal 26S rDNA analysis: Particularly suitable for yeast strain identification.

b. Fees

Services	Sample		Timeline
	Genomic DNA	Bacteria
6s rDNA Sequencing	$50 /sample (Cloning and sequencing)	$65/sample (Cloning and sequencing)	7 ~ 15 business days
	$33.5/sample (PCR products are sequenced)	$50/sample (PCR products are sequenced)
Fungal ITS Sequencing	$50 /sample (Cloning and sequencing)	$85/sample (Cloning and sequencing)	7 ~ 15 business days
	$33.5/sample (PCR products are sequenced)	$65/sample (PCR products are sequenced)
Fungal 18s rDNA Sequencing	$50 /sample (Cloning and sequencing)	$100/sample (Cloning and sequencing)	7 ~ 15 business days
	$33.5/sample (PCR products are sequenced)	$65/sample (PCR products are sequenced)
Fungal 26S rDNA D1 / D2 region Sequencing	$50 /sample (Cloning and sequencing)	$65/sample (Cloning and sequencing)	7 ~ 15 business days
	$33.5/sample (PCR products are sequenced)	$50/sample (PCR products are sequenced)

c.Results

All related data (including photos of gel analysis, NCBI reference strain identification results, etc.), sequencing trace files will be provided.

5.2 Microbial Flora analysis

Services	Sample		Timeline	Remarks
	Genomic DNA	Bacteria
RAPD	$8.5/sample	$25/sample	10 to 15 business days	/
Rep-PCR	$8.5/sample	$25/sample	15 to 20 business days	/
PCR-RFLP	$13.5/sample	$30/sample	15 to 20 business days	/
PCR-SSCP	$16.5/sample	$33.5/sample	15 to 20 business days	/
ARDRA	$83.5/sample (mixed bacteria)	$100/sample (mixed bacteria)	20 to 25 business days	Recovery and cloning of difference bands Sequencing charge of $80/clone
LARD	833.5 /sample(mixed bacteria)		20 to 25 business days	Including 50 positive clones Partial sequence analysis
DGGE	250 /sample(mixed bacteria)		25 to 30 business days	Recovery and cloning of difference bands Sequencing charge of $80/clone

c. Results

• The report, including material and methods, photo of gel analysis.
• Construction of RIS Library, sequencing trace files, the plasmid containing the gene and puncture bacterial.
• data analysis, including the NTSYS-pc software for fingerprint cluster analysis, phylogenetic tree constructed maps.

6.Suppression Subtractive hybridization

price and experimental period: 5835 dollar,45 working days

7.Single Nucleotide Polymorphism (SNP) Genotying Services

Typing methods	Quantity		Unit price		Timeline	Remarks
Mutation or SNP screening by Sanger sequencing	<=20 exons		$13.5/exon		10 business days	Charge of primers will be calculated case by case
	20~100 exons		$10 /exon		14 business days	>100, Inquire
Sequencing varification for known SNPs	<=20 sites		$10/site		10 business days	>100, Inquire
	20~100 sites		$8.5/site		14 business days
PCR-RFLP	<=500 sites		$2 /site		15 business days	>1,000, Inquire
	500~1,000 sites		$1.7/site		25 business days
TaqMan probe	<=500 sites		$3.5/site		20 business days	Charge of primers will be calculated case by case >1,000, Inquire
	500~1,000 sites		$3 /site		25 business days
Multiplex SNaPshot	The following price is for number of samples <=500					For samples>500, SNP site>20 per sample, Inquire
	<=2 sites per sample	$8.5 sample		30 business days
	3~10 sites per sample	$1.7~$3.5/site		30 business days
	11~20 sites per sample	$0.85~$1.7/site		30 business days
MALDI-TOF	The following price is for number of samples <=1000					For samples>1000, SNP site>20 per sample, Inquire
	<=4 sites per sample	$17 /sample		30 business days
	5~10 sites per sample	$1.5~2 /site		30 business days
	11~20 sites per sample	Inquire		Inquire
LDR	3~10 sites per sample	$1~2/site		15 business days		samples>100 charge of primer will be calculated case by case
	>11 sites per sample	<=$1/site		20 business days

8.Illumina fiber beads confocal analysis platform for high throughput gene

8.1 Illumina Fiber beads chip price of services

Type	Style	Price($)	Remarks
Whole-genome SNP genotyping chip	HumanCytoSNP-12 v1.0 DNA Analysis BeadChip Kit	416.7 /case	A multiple of 12 samples
	Human660W-Quad v1.0 DNA Analysis BeadChip (16)	933.5/case	A multiple of 16 samples
	Human1M-Duo v2.0 DNA Analysis Kit (8)	1466.7 /case	A multiple of 8 samples
	CanineSNP20 Canine whole genome SNP genotyping chip	666.7 /case	A multiple of 48 samples
	BovineSNP50 Bovine whole genome SNP genotyping chip	633.5 n/case	A multiple of 48 samples
	OvineSNP50 Sheep genome SNP genotyping chip	583.5 /case	A multiple of 48 samples
	PorcineSNP60 Porcine whole-genome SNP genotyping chip	583.5 /case	A multiple of 48 samples
Linkage analysis of SNP chip	Linkage Panel Linkage analysis of human chip	250 /case	A multiple of 96 samples
	Mouse LD Linkage Linkage analysis of low-density chips mice	250 /case	A multiple of 96 samples
	Mouse MD Linkage Chip density linkage analysis in mice	416.7/case	A multiple of 96 samples
Whole genome expression microarray	HumanRef-8 BeadChip Expression of the human genome chip	533.5 /case	Best of 8 samples
	Human-6 BeadChip Expression of the human genome chip	1083.5 /case	Best of 6 samples
	MouseRef-8 BeadChip Mouse whole genome expression chip	833.5 /case	Best of 8 samples
	Mouse-6 BeadChip Mouse whole genome expression chip	1083.5 /case	Best of 6 samples
	RatRef-8 BeadChip Rat whole genome expression chip	833.5 /case	Best of 12 samples
MicroRNA chip	Human microRNA expression profiling microarray	1041.7 /case	Best of 12 or 96 samples
	mouse microRNA expression profiling microarray	1041.7 /case
Methylation microarray	Human Methylation27 Methylation of the human genome chip	1041.7 /case	At least 12 samples
	Methylation Cancer Panel I Methylation in human cancer chip	833.5 /case

Type			Style					Sites		Price(dollar /case)			samples/ chip
Human genome SNP genotyping chip			Human Omni5-Quad (new)					5000,000		2,000			4 samples
			Human Omni2.5-8					2500,000		1333.5			8 samples
			Human Omni1-Quad					1140,000		916.7			4 samples
			Human OmniZhongHua-8 (new)					900,000		750			8 samples
			Human OmniExpress-12					700,000		666.7			12 samples
			Human CytoSNP-12					300,000		583.5			12 samples
			Human Exome-12(new)					250,000		366.7			12 samples
			Human Linkage-24					6056		533.5			24 samples
Animal plant genome SNP genotyping chip			Cow HD chip					70		800			8 samples
			Cow SNP50 chip					50,000		666.7			24 samples
			Cow LD chain chip					6000		366.7			24 samples
			Dog HD chip					170,000		800			12 samples
			Sheep SNP50 chip					50,000		666.7			12 samples
			Horse SNP50 chip					50,000		666.7			12 samples
			Pig SNP60 chip					60,000		666.7			12 samples
			Corn SNP50 chip					50,000		666.7			24 samples
Methylation detection chip			Human Methylation 27K					27,578		833.5			12 samples
			Human Methylation 450K					480,000		1133.5			12 samples
Microarray			HumanHT-12 V4					47,231		633.5			12 samples
			MouseWG-6					45,281		700			6 samples
Custom SNP chip(more than 480 samples)
Sites	96			144	192	384			768		1,152			1536
Price($)	4167			5,000	6700	11670			15300		20834			2250
Custom methylation chip(more than 480 samples)
Sites		96 methylation sites					384 methylation sites					other
Price($)		41700					100000					inquriy

9.Methylation Analysis

a.Services

Bisulfite modification and sequencing method (BSP)

Frommer et al (1992) proposed methods of DNA methylation analysis, this method is reliable and precise. procedures:

• Prepare genomic DNA of Sample
• Bisulfite treatment (including purification and de-sulfosalicylic reaction) to convert unmethylated cytosine to uracil, while methylated cytosine remains unchanged.
• Using methylation primer design software to design primers at the both ends of methylated regions.
• Fragment amplification and PCR product recovery.
• PCR products were sequenced directly or after cloned (quantitative analysis), analysis of each CpG island methylation.
• Data Statistics. unmethylated DNA samples are used as a control.

Methylation specific PCR method (Methylation-Specific PCR, MS-PCR)

Herman et al (1996) in the use of bisulfite treatment on the basis of the new methods. High sensitivity of this method is mainly used for qualitative research. Procedures:

• Prepare genomic DNA of Sample
• Bisulfite treatment (including the purification and de-sulfosalicylic reaction).
• Using methylation primer design software design methylation specific primers (primer 1) or non-methylated primers in methylated regions (primer 2) 2 pairs.
• For specific PCR amplification, only the fragments fully combinate with methylated or non-methylation specific primers can amplify the product.
• Detection of MSP amplification, if DNA methylation for the treatment chain primers amplified fragments, it shows that the detected methylation sites exist; if used for the treatment of non-methylated DNA chain The primers amplified fragments, then the detected methylation sites does not exist.

b.Fees

Services	Quantity	Unit price ($)	Timeline	Quantity	Price
Bisulfite modification and sequencing method	<=50	$110/sample/gene (offer sequencing results of 5 clones )	15 to 20 business days	>50	Inquire
		$165/sample/gene (offer sequencing results of 10 clones)	20 to 25 business days	>50	Inquire
methylation specific PCR method	<=100	$50/sample/gene	10 to 20 business days	>100	Inquire
Combined Bisulfite Restriction Analysis	<=100	$50/sample/site	15 to 20 business days	>100	Inquire
High Resolution Melting method	<=200	$45/sample/gene	15 to 20 business days	>200	Inquire
Pyrosequencing	<=200	$75/sample/gene	15 to 20 business days	>200	Inquire

Methylation detection chip	Sites of the amount of information	Unit price /sample ($)	The number of samples / chip	Timeline
Human Methylation 450K	480000	$1,135	12 samples	4 weeks
Customized methylation chip	96 methylation sites	Inquire		15 weeks
	384 methylation sites	Inquire		20 weeks

9.3 Results

Lab report (experimental steps, including the BSP method statistical results of 5 clones), PCR electrophoresis photographs, sequencing trace files.

10.Virus packaging

10.1 Adenovirus packaging services

10.2 Lentiviral packaging services

a. Service Quote

Services		Quantity	results	Price($)	Cycle
Vector construction	ShRNA vector of the target gene	1	Report	166.7	1 to 2 weeks
	Target gene shRNA vector Subscription	5	Report(4 object +1 negative contrast)	666.7	2 to 3 weeks
siRNA lentiviral screening	Buy cell recovery and training	1	Cell state Image	83.5	2 working days
	Slow virus infection experiments	1	The infection efficiency images and analysis	83.5	3 working days
	shRNA design	3	Target information	Free	1 working days
	shRNA vector construction	4	Vector Construction Report	666.7	2 to 3 weeks
	Lentiviral packaging (10e8TU/ml)	5	The virus liquid and the titer test report	1333.5	2 to 3 weeks
	The slower virus infection ( interference filter)	4	Picture	333.5	1 to 2 weeks
	qRT-PCR primer design and verification	2	Primer sequences ( containing an internal reference )	33.5	1 weeks
	qRT-PCR detect	3*5	Experimental data and analysis	300	1 to 2 weeks
	Western Blot detect	5	Experimental data and analysis ( primary antibody )	333.5	1 to 2 weeks
	Total price	/	/	3500	7 to 10 weeks
Establish stable Filter cell lines	The effective siRNA lentiviral repeated infection ( positive + negative )	2	Picture	266.7	1 to 2 weeks
	Stable cell strain was amplified ( positive + negative )	2	Cell picture	333.5	3 to 4 weeks
	qRT-PCR detect	3*2	Experimental data and analysis	200	1 to 2 weeks
	Western Blot detect	3*2	To provide an anti- experimental data analysis (customer )	200	1 to 2 weeks
	Infected cells (25T flasks , positive ( two kinds ) + negative , each bottle )		Cells and photo	333.5	1 to 2 weeks
	Total price	/	/	1333.5	6 to 9 weeks

11.RNA Interference Service

a.Fees

Services		Quantity	Results	Price ($)	Timeline
shRNA vector construction	Gene specific shRNA sequence is designed according to the target gene and cloned to shRNA expression vector	1	Report	$165	1 to 2 weeks
	Gene specific shRNA vector set	5	Report(4 gene specific shRNA vector +1 negative contrast)	$665	2 to 3 weeks
siRNA lentiviral screening	Cell culture	1	Image of cultured cells	$85	2 business days
	Lentiviral infection	1	Infection efficiency images and analysis	$85	3 business days
	shRNA design	3	Target information	Free	1 business days
	shRNA vector construction	4	Vector Construction Report	$665	2 to 3 weeks
	Lentiviral packaging (10e8TU/ml)	5	The virus liquid and the titer test report	$1330	2 to 3 weeks
	The Lentiviral infection ( interference filter)	4	Picture	$330	1 to 2 weeks
	qRT-PCR primer design and verification	2	Primer sequences ( containing an internal reference )	$30	1 weeks
	qRT-PCR detection	3*5	Experimental data and analysis	$300	1 to 2 weeks
	Western Blot detection	5	Experimental data and analysis ( primary antibody )	$330	1 to 2 weeks
	Total price	/	/	$3,500	7 to 10 weeks
Establish stable cell lines	The effective siRNA lentiviral repeated infection ( positive + negative control)	2	Picture	$265	1 to 2 weeks
	Stable cell strain was amplified ( positive + negative control)	2	Cell picture	$330	3 to 4 weeks
	qRT-PCR detection	3*2	Experimental data and analysis	$200	1 to 2 weeks
	Western Blot detection	3*2	To provide an anti- experimental data analysis (customer )	$200	1 to 2 weeks
	Infected cells (25T flasks , positive ( two kinds ) + negative , each bottle )		Cells and photo	$330	1 to 2 weeks
	Total price	/	/	$1,330	6 to 9 weeks

b.Results

Reports include: siRNA sequences, construct vectors sequencing report, quantitative PCR test report, Western Blot image and all the experimental data and detailed experimental procedures