1. Drop off your sample(s) in a ziplock bag along with the online order number at any one of our convenient dropboxes (if located on onsite at your institution or campus).
2. If no ACGT dropbox is located at your campus or institution, and you will be shipping your samples, please send to:
35 Waltz Drive
Wheeling, IL 60090
ACGT accepts samples Monday through Friday from the U.S. Postal Service and all other courier delivery services (e.g. UPS, DHL, FedEx, etc.). ACGT accepts samples on Saturday from FedEx only. To ensure your samples are received on Saturday, please mark “Saturday Delivery” on the FedEx Airbill. Please make sure to indicate the online order number along with the shipment. The processing of samples without a corresponding order number will result in delay.
3. DNA samples in tubes may be shipped at room temperature for overnight delivery. It is not required to send the samples on dry ice or wet ice.
4. Template DNA stored in individual tubes should be capped tightly to prevent accidental spillage or cross contamination.
5. DNA shipped as a pellet can be also shipped at room temperature.
6. Use adequate padding to prevent any damage during shipment. ACGT provides bubble bags free of charge found at any of our dropboxes. If an ACGT dropbox is not found at your campus or institution, you may request bubble bags from ACGT Customer Service at 1-800-557-2248 or [email protected].
DNA Samples in 96-Well Plate
1. 96-well plates should be sealed properly to prevent cross-contamination or evaporation during shipment. ACGT recommends using strip caps to seal plates with a Parafilm wrap to avoid spillage and cross-contamination. High quality heat seals are also recommended.
2. ACGT recommends freezing your plate samples and shipping with dry ice. You may pack the plates (while still frozen on dry ice) in a Styrofoam box. Please ensure there is a sufficiency of dry ice on the top, bottom, and on both sides of the plate(s).
3. If shipping more than one plate, wrap the plates in aluminum foil and/or vinyl tape (not lab tape) so your plates will not shift or move around during shipment. Lab tape tends to become brittle and rip apart when exposed to dry ice.
Bacterial Agar Plates
1. Use a Parafilm wrap on the edges of the plates and place them in the container with the lid side down. Pack with a protective bubble pad.
2. Plates can be shipped at room temperature if not overgrown.
Bacterial Agar Lawns
1. Use a Parafilm wrap on the edges of the lawns.
2. Pack agar lawns in a Styrofoam cooler. Plates should be cooled to room temperature to prevent condensation. Once the plates have cooled, place them in the cooler with the lid side down.
3. Place ice packs in the cooler. Do not place ice packs directly on plates as condensation may leak into the plates. Clearly indicate “THIS END UP” with arrows for a proper handling and delivery.
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 in a single tube:
AMPure PCR Purification Kit from Agencourt (PDF)
QIAquick PCR Purification Kit from Qiagen (PDF)
Zymoclean Gel DNA Recovery Kit from Zymo Research (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)
Precautions with Qiagen Plasmid Kit
1. 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.
2. Use the recommended quantity of LB (not Terrific broth) for cell growth.
3. Wash the DNA pellet at least once as directed with 70% ethanol.
4. 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.
5. Dry the template DNA completely. Residual ethanol is detrimental to Taq cycle sequencing resulting in data with a drastically reduced signal.
6. To remove residual ethanol, dry the DNA for 5 min. in a properly operating speedvac.
7. If air-drying, make sure that thd DNA is dry(no fluid in the tube, the DNA pellet does not look wet.)
8. When air drying, a brief 15 min incubation of the open tube at 65°C is often sufficient to completely dry the DNA.
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 OD260/230 of about 2.2.2. Low 260/280 indicates protein contamination, high OD260/280 ration indicates presence of organics contamination.
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.
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.
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.
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:
1. Perfect matches
2. No alternative hybridization sites in template
3. No palindromic sequence present, particularly at the 3’ end of primer
4. Tm of ~56-62°C. Avoid low Tm (i.e. 40-45°C) If Tm is low, make the primer longer.
5. Length of primer should be 18-23bpc.
6. GC “clamp” on the 3′ end.
7. Desirable [GC] = ~50-55%
8. Avoid strings of four or more of the same base if possible.
Host Bacterial Strains
The host strain used for a specific template preparation can impact template quality. Please note the following.
1. DH5 alpha host strains consistently produce good results.
2. 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.
3. JM101 (JM 100 series) is not recommended.
4. Avoid Terrific broth and other rich media.
5. Avoid host strains TG1 and TG2 which contain high carbohydrate levels.