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Located in rooms
B065 and B017
bklogo

 

Pyrosequencing
 

Frequently Asked Questions
  1. Where is the PAN Facility located/contact info?
  2. What is the turnaround time?
  3. How much does it cost?
  4. What primers does the PAN Facility provide?
  5. What concentration should the DNA be?
  6. What concentration should the primers be?
  7. How do I get my results?
  8. Why do I need three primers?



1. Where is the PAN Facility located/contact info? (top)
We are located in the basement of the Beckman Center, B017.  We also have a drop-off area in cold room B024.

Phone: (650) 723-3189
Email: alberto5@stanford.edu

Shipping Address:        
Pyrosequencing/PAN Facility
Beckman Center, B017
279 Campus Drive, West
Stanford, Ca 94305


2. What is the turnaroud time?
(top)
Turnaround time is project dependent. On average assay design take a week. Optimizing PCR can take a week. Once optimal PCR conditions have been established, pyrosequencing samples are processed in ~two days.


3. How much does it cost?
(top)
Please check our current price list.


4. What primers does the PAN Facility provide?
(top)
Unfortunately, the PAN Facility does not provide any primers for Pyrosequencing. We are working on establishing a universal PCR primer set which we hope will be available in the near future.


5. What concentration should the DNA be?
(top)
In general, it is best to follow the your PCR kit's manufacturer's guidelines. Typically, we have seen good results with 10ng - 20ng of total DNA per PCR rxn.


6. What concentration should the primers be?
(top)
Both PCR and Pyro-Sequencing primers should be at an initial concentration of 5uM. Consider supplying 5ul of each primer for each rxn.


7. How do I get my results?
(top)
Results are emailed to you, and can be viewed using Qiagen's PyroMark software. You can obtain a free copy of the software by contacting Alberto Lovell at 650-723-3189.


8. Why do I need three primers?
(top)
You need at least two primers for PCR. One PCR primer MUST BE biotinylated on the 5' end. The biotinlyated PCR primer can be either the forward or reverse PCR primer, that's up to you. The biotinlyated PCR strand is captured/purified prior to pyrosequencing. A pyro-sequencing primer (the third primer) is added to the purified biotinlylated PCR strand and pyrosequencing commenses. The pryo-sequencing primer MUST BE in the opposite direction of the biotinlyated primer. You can use the non-biotinlyated PCR primer as your pyro-sequencing primer, but please be aware that this strategy is not as efficent as using a nested pyro-sequencing primer.