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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:

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 reoptimize the PCR to obtain a single product.

Template Quantity:

Quantitate DNA by Spectrophotometer (Nanodropor equivqlent). 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. 

*For DNA purification we suggest using BIONEER purification kits

Type of DNA	Amount	Concentration
DS Plasmid DNA (3Kb-10Kb)	12 μl/reaction	100-300 ng/μl
PCR Fragments>400bp	12 μ/reaction	30-80 ng/μl
PCR Fragments<400bp	12 μl/reaction	10-20 ng/μl
BAC DNA	12 μl/reaction	0.5-1 μg/μl
Cosmid DNA	12 μl/reaction	0.5-1 μg/μl
lambda DNA	12 μl/reaction	0.5-1 μg/μl

 Primers

• Primer concentration:   5 pmol/μl (10pmol/reaction).
• Universal primers can be provided by hylabs (please check our primer list)
• Your Own Primer - guidelines for optimal prime design:

What not to send

• Samples with high salt concentration (low 260/230 ratio).
• 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 (JM 100 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 can not be avoided an additional ethanol precipitation is recommended.

Effect of Too Little Template;  Too little 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 resuspend the DNA in TE. Use ddH20 or 10 mM Tris pH 8.5.
• If using a column-based kit, insure that all of the EtOH is evaporated before eluting the DNA.
• If concentrating the DNA insure 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.

 Frequent reasons 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:

1. The 3730 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.
2. Too little template results in reactions with little or no signal and poor or no base calling.
3. Too much DNA produces reactions that terminate prematurely, often with fewer than 250 bases of reliable sequence data.
4. Poor quality template DNA. Template DNA must be free of residual ethanol and salt.
5. Insufficient primer concentration or poor quality.
6. The Tm of the primer is << 50 C.
7. The template does not contain a sequence complementary to the primer.
8. Primer and/or template was not added to the reaction.
9. Using the same primers for sequencing as were used for PCR.
10. 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.
11. 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 core DNA Sequencing Facility is open 5 days/week. Please call us if you need any help from our sequencing technicians between 8:00-12:00 and 15:30-17:00.

For questions or suggestions please contact us at:   lab1@hylabs.co.il

                                                                      08-9366475 ext: 222