GUIDELINES FOR DNA SAMPLE PREPARATION
We understand that every sequencing result and turnaround time is critically important for you to proceed to the next step of your research. In order to generate quality sequencing data, please follow the instructions below to prepare your DNA samples.
Template DNA Purification Kit
The quality of the template DNA directly affects the quality of the DNA sequencing results. We recommend using one of the following methods:
Plasmid DNA purification system in a single tube:
QIAprep Spin Miniprep Kit from Qiagen (PDF)
Wizard® Plus SV Minipreps DNA Purification Systems from Promega (PDF)
PurElute™ Plasmid Miniprep Kit from Edge BioSystems (PDF)
Quantum Prep®Plasmid Miniprep Kit from Bio-Rad (PDF)
Plasmid DNA purification system for 96-well plate:
QIAprep 96 Turbo Miniprep Kit from Qiagene (PDF)
Montage® Plasmid Miniprep96 Kit from Millipore (PDF)
SeqPrep™ 96 Plasmid Prep Kit from Edge BioSystems (PDF)
PCR DNA purification system for 96-well plate:
QIAquick 96 PCR Purification Kit from Qiagen (PDF)
ExcelaPure 96-Well UF PCR Purification Kit from Edge BioSystems (PDF)
PCR Amplification Products
It is essential that the DNA is free of contaminants, unused primers or dNTPs. PCR templates that do not undergo any kind of post PCR clean up are not suitable for sequencing and will yield unusable sequence data. It is highly recommended that your PCR template is first checked on a gel to confirm that there is a specific product with the correct size.
The EXO/SAP protocol to clean the PCR amplicon frequently damages the PCR primer attachment sites. We recommend that internal primers are used for sequencing because it lowers the risk of poor sequencing results due to the damaged PCR primer attachment site.
We recommend using gel electrophoresis, where the band intensity of a sample DNA is compared to a standard ladder, as the most accurate quantitation. For example, the 1.6 kb band of the invitrogene’s 1 KB plus ladder contains 8% of total amounts of DNA presents in the ladder mixture, and can be used as quick reference for estimating the concentration of your template.
We also recommend using Nanodrop Spectrophotometer that measures 1 µl samples with high accuracy and reproducibility.
Host Bacterial Strains
The host strain used for a specific template preparation can impact template quality. Please note the following.
- DH5 alpha host strains consistently produce good results.
- HB101, MV1190, JM109 and XL1 Blue host strains show some variability in result quality. XL1 Blue grows slower than most strains and can lead to decreased DNA yields and it does not respond well to TB as other strains.
- 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.
ACGT, Inc. provides 50 different free-of-charge universal primers. Specific internal primers can be ordered through ACGT, Inc. To design your own primers, please follow these considerations:
- Perfect matches
- No alternative hybridization sites in template
- No palindromic sequence present, particularly at the 3’ end of primer
- Tm of ~56-62°C. Avoid low Tm (i.e. 40-45°C) If Tm is low, make the primer longer.
- Length of primer should be 18-23bpc.
- GC “clamp” on the 3′ end.
- Desirable [GC] = ~50-55%
- Avoid strings of four or more of the same base if possible.
DNA in General
- It is critical that the plasmid template is free of any contaminants including buffers, salts, organics, and proteins.
- Submit DNA in deionized water, not in TE. Buffer components inhibit the sequencing reaction and cause failed runs.
- If possible, use of phenol or chloroform during the purification procedures should be avoided. An additional ethanol precipitation is recommended if phenol or chloroform is used.
- DNA should give an OD260/280 of between 1.7-1.9 and an OD260/230 of about 2.2.2. Low 260/280 indicates protein contamination, high OD260/280 ration indicates presence of organics contamination.
- If your PCR amplification procedure generates a product with a single robust band, column purification will suffice. If it generates multiple bands, gel purification of the band with the desired size is required.
Precautions with Qiagen Plasmid Kit
- Do not overload the Qiagen resin. Usually, a poor yield of plasmid DNA results, presumably due to competition with RNA fragments for binding to the Qiagen resin.
- Use the recommended quantity of LB (not Terrific broth) for cell growth.
- Wash the DNA pellet at least once as directed with 70% ethanol.
- Residual salt in the final template will interfere with the activity of Taq polymerase resulting in sequence data which extends fewer than 200 bases from the primer and exhibits a low signal to noise ratio.
- Dry the template DNA completely. Residual ethanol is detrimental to Taq cycle sequencing resulting in data with a drastically reduced signal.
- To remove residual ethanol, dry the DNA for 5 min. in a properly operating speedvac.
- If air-drying, make sure that thd DNA is dry(no fluid in the tube, the DNA pellet does not look wet.)
- When air drying, a brief 15 min incubation of the open tube at 65°C is often sufficient to completely dry the DNA.