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 - Wizard® Plus SV Minipreps DNA Purification       Systems from Promega - PurElute™ Plasmid Miniprep Kit from Edge       BioSystems - Quantum Prep®Plasmid Miniprep Kit from Bio-Rad  Plasmid DNA purification system for 96-well plate: - QIAprep 96 Turbo Miniprep Kit from Qiagene - Montage® Plasmid Miniprep96 Kit from Millipore - SeqPrep™ 96 Plasmid Prep Kit from Edge       BioSystems  PCR DNA purification system in a single tube: - AMPure PCR Purification Kit from Agencourt - QIAquick PCR Purification Kit from Qiagen - Zymoclean Gel DNA Recovery Kit from Zymo       Research  PCR DNA purification system for 96-well plate: - QIAquick 96 PCR Purification Kit from Qiagen - ExcelaPure 96-Well UF PCR Purification Kit from       Edge BioSystems  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 unuseable 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.  Quantitation 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   decresed DNA yields and it does not respond to TB   as do other strains. - JM101 (JM 100 series) is not recommended. - Avoid Terrifir broth and other rich media. - Avoid host strains TG1 and TG2 which contain high   carbohydrate levels.

Sequencing Primers 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 - Appropriate length to give Tm of ~55-62°C. Avoid   low Tm (i.e. 40-45°C) If Tm is low, make the primer   longer. - 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 1. It is critical that the plasmid template is free of any contaminants including buffers, salts, organics, and proteins. 2. Submit DNA in deionized water, not in TE.  Buffer components inhibit the sequencing reaction and cause failed runs. 3. 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. 4. DNA should give an OD260/280 of between 1.7-1.9 and an OD200/260 of about 1.1. Low 260/280 indicates protein contamination, high OD260/280 indicates possible RNA or residual organics contamination. High OD200/260 indicates contamination by organics salts. 5. 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.