Sequencing Sample Submission
- A fee of $5.00 is charged for each REACTION (template/ primer combination). We do not charge by the base.
- We use a MINIMUM of 10 ng template for PCR product sequencing and 50 ng for plasmid sequencing, and 50 ng for single strand DNA sequencing. Please make sure that you supply us with the appropriate amount of template for the number of reactions you are requesting.
- We require a minimum concentration of 5 ng/ul for PCR products, 50 ng/ul for plasmids, and 15 ng/ul for single strand DNA. Should the concentration of your template fall below these levels, you are responsible for concentrating the sample by the method of your choice. The concentration of the sample should be determined again following any procedure.
- A WORKORDER sheet is required for every template, but the same sheet may be used if you have multiple templates using the same primer(s).
Make sure you include the following information:
- Your name, phone number, Department address, and e-mail address.
- A current budget number, which must be supplied at the time the workorder is submitted. No exceptions will be allowed.
- Template information:
- Name of the template (eight characters or less)
- Type of DNA, plasmid or PCR amplified
- Vector and insert length
- Sample concentration, solvent, and quantity. Please write the concentration in terms of ug/ml, ug/ul, or ng/ul, NOT in terms of the total volume, such as 5.2 ug/320 ml.
DO NOT "guess" the concentration of your sample from an agarose gel. We have found large discrepancies between estimated values and concentrations determined on a spectrophotometer. Our protocols rely on spectrophotometric values.
DNA should be in water. DO NOT use TE or Tris buffer, because they could interfere with the sequencing reaction.
- Sample-purification history (the type of protocol is sufficient)
- The primer(s) you wish to use, including the primer sequence, length, and concentration. We maintain some standard sequencing PRIMERS in the lab, so it is not necessary to supply us with these. If you have a specific primer that you use frequently for sequencing, we will keep a stock of it in the lab for you.
- Once a sample has been submitted to the Sequencing Facility, we will perform all sequencing reactions and load them on the ABI 3730 capillary sequencer. Typically you will have data 1-2 days after submitting the sample. This can vary depending on the number of sequencing samples, and whether Genescan (fragment analysis) is run that week.
- Labeling Template Microfuge Tubes
Label each sample with the following:
- Your last name(on side of tube).
- The name of your template. Each name should be limited to eight or fewer characters. (THIS INFORMATION MUST BE ON TOP OF TUBE)
- The concentration of your template. Please write the concentration in terms of ug/ml, ug/ul, or ng/ul, NOT in terms of the total volume, such as 5.2 ug/320 ml(on side of tube).
- Your major professor's last name (where applicable on side of tube).
- Labeling Primer Microfuge Tubes
Label each with the following:
- The name of the primer, limited to six or fewer characters(on cap and side of tube).
- The concentration of the primer. Please write the concentration in terms of ug/ml, ug/ul, or ng/ul, NOT in terms of the total volume, such as 5.2 ug/320 ml (on side of tube).
- Your major professor's last name (where applicable on side of tube)
Common Primers Stocked in the Sequencing Facility
|NAME||SEQUENCE (5' TO 3')|
|M13 for||CGT TGT AAA ACG ACG GCC AG|
|M13 rev||CAG GAA ACA GCT ATG AC|
|T3||ATT AAC CCT CAC TAA AGG GA|
|T7||TAA TAC GAC TCA CTA TAG GG|
|SP6||GAT TTA GGT GAC ACT ATA G|
|*P8||CCT CTA GAA ATA ATT|
|*P9||TTC GGG CTT TGT TAG CAG|
|*T7 terminator||GCT AGT TAT TGC TCA GCG G|
* Used for sequencing into the multiple cloning site of some PET vectors