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Frequently Asked Questions

1. Submitting Online Orders

Q: Why was my on-line order not processed correctly?

A: Please check the following.

  • PO number is ready and entered.
  • No strange characters or spaces were entered in the names of your DNA samples and primers when placing your order.
  • Was there a high-traffic moment on the web?  Try connecting again.

Q: There is a discrepancy between the number of samples I am submitting and the number of samples indicated in the database. How do I fix this?

A: The database can sometimes incorrectly indicate the number of samples in an order. The database should indicate the number of samples (if processed correctly). If not, there might be a delay in processing your samples and you should NOT click "submit."

Please do not try to delete samples from the sample sheet directly.  In addition, if you make a mistake in placing your order, please delete the order and start over -- the best way to get the samples added correctly so that sequencing can proceed without complications.


2. Optimizing DNA Sequencing

Q: Why didn't my DNA samples get a higher quality score (higher quality sequence)?

A: There are several different reasons that a DNA sequence sample fails to produce quality sequence. Please let us know if you would like us to re-run your samples if the quality is not what was expected. However, the solution is often in re-doing the sample preparation.

Here's a list of things to consider as you think about improving DNA sequence quality.

For the DNA,

  • Too high or too low in concentration?
  • Correct total volume was added to the plate well?
  • OD 260/280 ratio is close to 1.8?

Other considerations:

  • Is there EDTA in the sample?
  • For GC-rich sequences, did you indicate "DMSO" on the sample submission form?
  • Does the DNA contain secondary structures? If so, did you indicate "higher annealing" on the sample submission form or have a chat with Sequencing personnel about annealing temps for your sample and primers?
  • Are there long stretches of the same base (homopolymer stretch) or repeats that could cause the polymerase to "slip"?
  • Did you include the correct primers?
  • Are the primers at least 18 base-pairs long and have a Tm between 55 and 65?  About 50% GC?
  • Was your bacterial host strain (for plasmid prepped DNA) appropriate (DH5-alpha and HB101 work best; MV1190 and XL1 yield variable results; JM101 yields poor results).

If you still have questions, please contact Li Chan to discuss ways to optimize your sequencing reactions.

 

Troubleshooting problems with DNA sequencing

Common causes of sequencing failures:

Too much or too little template DNA.

Click here to read our guidelines on DNA sample preparation and DNA concentration.

Another common problem is high GC content.

Please be sure to let us know if you expect your DNA samples to have higher than average GC content. We will make adjustments to the reaction mix (such as the addition of DMSO) to help overcome problems created by high GC content.

Hairpins -- such as those in shRNA plasmid constructs -- can also be problematic.

Our staff are expert at adjusting sequencing conditions to sequence over hairpins and has experience sequencing shRNA constructs. Please let us know if you are submitting sequences that you know have hairpins or other potentially structure-forming sequences.

Click here to read our FAQs.

Still having trouble? Please contact Li Chan at (617)432-7708.

 

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