Process
All samples sent to the MBRF for sequencing should be supplied in H20. Please, do not submit your samples in TE buffer. The EDTA will inhibit the Big Dye polymerase’s effectiveness. We like all samples to be measured here in the lab using one of our Hoefer Fluorometers. It is much more accurate for our purposes of sequencing. The lab provides gloves, tips, and buffer supplies as well as directions on how to use the fluorometers. There is no charge for you measuring your samples or using our supplies. However, if you like, we can measure your samples for you for a $1.00 charge per sample. Plasmid DNA should measure a minimum of 35ng/uL for us to sequence and at least 15uL’s at that concentration for each sequencing reaction requested. We have two “speed-vacs” available here in the lab for you to use if necessary. All primers should be brought in separate tubes at 5uM concentration. Again, keep the EDTA down to a minimum in your primers.
For PCR products, 10ngs DNA per 100 base pairs is the minimum we require for sequencing.
As a general rule, we like to use 10ng of template DNA per 100 bases in size. So, for a 4.5kb plasmid, we like to use 450ng per reaction. We use 1uL of 5uM primer per each reaction. We like to use 2uL of primer whenever possible at this concentration. The maximum reaction volume that we use is 15uL….but only when necessary. We like to perform our reactions using 12uL total reaction volume. In each reaction, we will add ABI’s Big Dye v3.1 Cycle Sequencing Mix along with 5x buffer that is supplied in the kit, then the remaining volume using H2O. We perform the actual sequencing reaction using ABI’s 9800 Fast Thermal Cycler. The more cycles you run, the greater the intensity of your product, and the lower the “dye blob” will be in your results. Removing any unused dideoxy-labeled nucleotides (purification) is done by using Sephedex G50 fine size exclusion columns. A good recipe for the G50 slurry can be found here at the lab. We are having excellent results using this protocol.
When sequencing reactions fail, we first try to analyze why they failed, and then will repeat the sequencing reaction upon request. Our policy on repeated reactions is that if we repeat using the same sample tube and primers provided, and they work the second time, then the repeat is free. However, since we are a cost recovery facility, someone must be responsible for the repeated reactions that fail a second time. We believe that responsibility fairly lies with the client.