Sample Preparation for DNA Sequencing

General Information

The MBRS aims for a turnaround time of 24 hours or less for the sequencing samples. Much of our cycle sequencing process is automated and the speed at which we can cycle, load, and run samples is dependent on how the samples are presented to us. We try to work within constraints of the computer systems and our robotic liquid handling system. File names, sample size, and quality are important. If we have to work with individual samples that do not conform, it takes longer for us to do the sequencing. All samples we receive by 10:30 AM will be processed that day and with some of that data being posted before 5:00 PM. However we process samples whenever we have a sufficient numbers to fill a run (48). Thus it is best to deliver your samples as soon as they are ready. It is not uncommon for us to start processing samples as early as 6:00 AM or as late as 4:30 PM. The faster samples get delivered to us the faster we get the results back.

Template and Primer

As noted MBSR prefers to perform the sequencing chemistry for you. Prepare your samples in the following manner: combine each sample template and its primer in a single 1.5 ml mircocentrafuge tube in the concentrations as indicated in the information below. Final sample volume should be 20 microliters. If they are not 20 ul in volume , add Milli-Q water to bring them to volume.

Use 3 to 5 pmoles of primer for all your reactions except if sequencing genomic DNA then use 30 to 50 pmoles of primer.

 

The MBRS does keep some common primers in the lab. These include, T3, Sp6, T7 universal, T7 terminator. M13, M13-40, and M13 reverse. You can request that we use our primers for your samples but we do charge extra because of the additional labor and additional controls that are run. For this reason we recommend you buy your own primers and add them to the sequencing reaction yourself.

DNA template should be quantified by measuring absorbance at 260nm/280nm. The higher the quality of DNA the better the sequencing results tend to be. 260/280 ratios are best if above 1.8. Remember crap in carp out.

Template type     Quantity

PCR Products:
100-200bp           5-10ng
200-500bp          10-20ng
500-1000bp        20-30ng
1000-2000bp      30-40ng
>2000bp             40-50ng

Single stranded 25-50ng
Double stranded 250-500ng
Cosmid, BAC 0.5-1.ug
Bacterial genomic DNA 2-3ug

 

Delivering samples to the Molecular Biology Shared Resource Lab.

Bring a copy of your order form with your sample(s). They 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).

We do offer reduced pricing for samples submitted in a 96 well-plate for sequencing. The set up for the samples is different than described above so please contact the Core and allow us to assist you.