Sample Preparation for DNA Sequencing
We aim for a turnaround time of 24 hours or less. Much of the cycle sequencing process is automated and the speed at which we can cycle, load, and run samples depend on how the samples are presented to us. We need to work within constraints of the computer systems and of the robotic liquid handling system. File names and sample size are the most important. If we have to work with individual samples that do not conform, it takes longer for everyone to get their data and cost to process a sample goes up.
The Molecular Biology and Proteomics Core has switched to a new electronic sample database management system. This system makes it easier to submit samples while giving the user automated data delivery, direct access to some older data, sample billing information, etc. An important feature of this system is that it will assign a sample number to each of the samples you submit. For the system to work correctly you will need use the assigned numbers on the sample tubes you submit. See these instructions for details.
Delivering Template and Primer to the Core Facility
A copy of your order form with your sample(s) can be dropped off 24/7 in refrigerators located in the laboratory of 241 Remsen, or the Borwell loading dock (next to the Rubin elevator).
Template and Primer
Core Facility prefers to do the sequencing chemistry. Prepare your samples in the following manner: mix each sample template and primer in a single tube 1.5 ml mircocentrafuge tube in the proportions indicated in the table below. The final sample volume should be 20 microliters. If they are lower, add Milli-Q water to bring them to volume. The liquid handling equipment works best with 20ul sample. If the sample is less than 20ul your template to primer ratio may be off and your data could be compromised. The Core Facility performs all cycling and cleanup operations needed for sequencing.
|Type of Template||Amount||Concentration||Volume|
|BigDye Terminator Mix||4ul|
|Double stranded template||200-500 ng||Not to exceed 10.8ul|
|Single stranded template||50-100 ng||Not to exceed 10.8ul|
|PCR Product 100-200 bp||1-3 ng||Not to exceed 10.8ul|
|PCR Product 200-500 bp||3-10 ng||Not to exceed 10.8ul|
|PCR Product 500-1000 bp||5-20 ng||Not to exceed 10.8ul|
|PCR Product 1000-2000 bp||10-40 ng||Not to exceed 10.8ul|
|PCR Product 2000 bp||40-100 ng||Not to exceed 10.8ul|
|cosmid, BAC||0.5-1.0 ugm||3.2ul|
Preparing the sample yourself
If you need to run specialized cycling reactions that we are currently not performing but still need us to do the sequencing please contact us. We can help you make sure you are performing reactions and clean up steps for optimal results. For instance the purification of the extension products is very important. If the unincorporated dye terminators are not thoroughly removed from the reaction, your sequence will be rendered unreadable due to the overwhelming fluorescence of the aggregated dye terminators. There are several acceptable methods for removing the unincorporated dye terminators. The cheapest way is ethanol precipitation. However, we have found this to be subject to a great deal of variability and do not recommend it. We recommend spin column purification of extension products. The Remsen stock room carries Edge BioSystems columns. These columns are relatively inexpensive and give good results on the average sample. However, we have observed some batch-to-batch variation. There are other methods available, such as magnetic bead technology and a membrane filtration method. However, they are designed for high throughput 96-well plate technology and would be of little use for small numbers of samples.
Submitting 96 well-plate
If you intend to do full 96 well-plate sequencing, please visit the Core and allow us to assist you in setting it up. There is a discount for a full plate of completed samples or samples that need to be cycled and cleaned but there are specific guidelines you need to follow.