Instructions for real time submissions

PLEASE NOTE: Due to recent events and the continuing concerns posed by potential bioterrorism agents, we have modified our online submission form to require that you provide us with information on the original biological source of any DNA that you will be asking us to obtain sequence from. "Original biological source" refers to the organism from which the DNA was first found in nature (human, HIV, Staphylococcus aureus, etc.). If you simply put a nonspecific term such as "E. coli clone" or something similar, this will not be sufficient. Further, we will not be able to accept DNA sequencing orders for select agents, until our legal responsibilities are clarified. Our current understanding is that, in order to receive select agent DNAs from you for sequencing, we would need to have a select agent permit, which we do not possess. We realize that adding yet another field to fill out on these forms can be tiresome, but this information is necessary to protect our staff against potential legal difficulties. Thank you very much for your understanding.

            - David W. Dyer, Ph.D.

 

 Instructions for Real Time submissions

THE FOLLOWING REQUIREMENTS MUST BE MET BEFORE YOUR SAMPLES CAN BE SUBMITTED Preparation of samples:

1. All samples must be submitted in 96-well format (V-bottom plates, skirted or non-skirted PCR plate, or Optical Fast plate).
2. If you are submitting <96 samples, then load the plate in column format (i.e. A1-H1, A2-H2 etc.) Partial plates can be reused for submission, in which case you can use the first available column (i.e. starting with A4-H4 etc.) Just be sure that if you start with A4 on your plate you also begin with A4 on the submission form.
3. Follow Applied Biosystems instructions according to what type of reaction you are doing. Each well should contain DNA, primer.

Example: (SYBR Green reactions - 25µl reaction)
cDNA template should come to a final volume of 9.5µl
Primer = 3 µl (1.5 µl forward primer + 1.5 µl reverse primer each @ 100µM concentration)

*Each set of reactions should be accompanied by comparable -RT rxns and NTC (no template control) reactions.
**Template and primer concentration will vary depending upon what your reaction specifications are. We find that the primer concentration listed above works with good efficiency for SYBR Green reactions.
***Applied Biosystems recommends 50 µl reactions, but we find that 25 µl is sufficient. If you prefer a larger reaction volume, please indicate this on the submission forms. For all TaqMan reactions, we will set up 50µl reaction unless otherwise told to do so.
****Only one type of reaction can be run on each plate. For example, we cannot run half a plate of SYBR Green and half TaqMan. The TaqMan runs in fast mode, the SYBR green does not. The same goes for two-step and one-step RT-PCR.
4. An SUR# (OUHSC) or PO# (off-campus) must be given at the time of submission.
For samples that need to be shipped, please dry down your template and primer mix in a PCR plate before sending to reduce the chances of sample loss during shipping.

Shipping Address:

The Laboratory for Genomics and Bioinformatics OUHSC
Attn: Mandy Gipson
975 NE 10th Street, BRC1106
Oklahoma City, OK 73104


5. A submission form must be filled out completely and included with your samples. An online submission form is in development.
   
   Definitions of terms on the form:
   
   Target: The nucleic acid sequence that you are studying.
   Calibrator: The sample used as the basis for comparative results.
   Endogenous control: A gene present at a consistent expression level in all experimental samples.
   
   -Each sample type requires and endogenous control.
   -If samples are spread across multiple plates, each plate must have an endogenous control. Additionally every plate must include an endogenous control for every sample type on the plate.

 TO KNOW

1. Turnaround time for reactions are dependent on the number of samples we receive each day. Customers can expect their data within a maximum of 48 hours from the end of the day that the samples are received (not including weekends, or holidays). For example, samples that are received on Monday by 4pm will be ready no later than 5pm on Wednesday. Larger orders of multiple plates may take longer than 48 hours to complete, but data will be made available as each individual plate is processed.
2. Finished data will eventually be accessible on the facility's website. In the interim, the data will be sent to the customer via email.
3. Samples are named automatically according to the well position in which it was submitted. (Example: the sample in well A1 will be named "a01")
   If you submit plates containing gaps between samples within a column you will be charged the regular real time PCR rate for these gaps.
4. Finished reactions will be named as follows:
date of run+run#_well#
example: 2002062101_A01
- - [note: the date is set up as (YYYY/MM/DD)]
5. Once the samples have been run, leftover templates and primers should be picked up within 2 weeks.
6. If you would like to pick up partial plates for re-use, please indicate in the special instructions of your order that you would like your plate saved for pickup. Remember that when plates are submitted with only certain parts of the plate covered, water may collect in uncovered wells during freezer storage. Reuse those plates at your own risk. We ask that people only take back plates that were submitted by them. We cannot be held responsible for lost or stolen plates.
7. If you have any additional needs or questions, please contact Mandy Gipson (405-271-2337) or Dr. Allison Gillaspy (405-271-1201 ext. 2).

 SUBMISSION FORMS

               real time submission form (an online submission forms is in development)