Frequently Asked Questions
How should I prepare my plasmid template?
Our customers have seen the best results with Qiagen.
However, several other vendors provide high quality plasmid prep kits.
If you already have a system in place, save the trouble and try your
own method FIRST before developing a new protocol. Make sure samples
are suspended in water and SALT-FREE!!
(back
to top)
How should I purify my plasmid template?
The Qiagen kit noted above elutes samples in water
ready to sequence. Other kits use salt buffers and require a final ethanol
precipitation and resuspension in water. We strongly discourage gel
purification. However, we do recommend that you visualize all template
preps by gel electrophoresis to ensure quality.
(back
to top)
How much plasmid template should I use?
Quantitate template using a spectrophotometer. Typical
reactions use 50ng per Kb of template, insert and vector combined. BE
PREPARED TO OPTIMIZE!!
Example: A reaction containing a 5.5Kb plasmid with a 1.5Kb insert requires
350ng template. (back to top)
How much primer should I use?
Standard reactions require 2 pmol of desalted primer.
Occasionally more primer is required for poorly annealing oligos or
exceptionally large plasmids, i.e. 20Kb+. Any number of vendors supply
high quality oligos. We typically use Gibco.
(back
to top)
How should I design my primer?
Use a primer design program such as Primer3. Be sure
to leave at least 50 bases between the primer and the target sequence.
(back to top)
Can I sequence directly from PCR product?
Yes, a well-optimized PCR reaction can be used directly
in a sequencing reaction. Purification by ethanol precipitation or spin
column can provide enhanced results, but is often unnecessary. We strongly
recommend designing a third, internal sequencing primer. External sequencing
primers from the original PCR reaction may work unreliably.
(back
to top)
How much PCR template should I use?
Ideal PCR template quantity for sequencing reactions
can be difficult to predict. As a general procedure: We run 10ul reactions
and visualize half on agarose to check template quality. The remaining
product we dilute 1:10 with water and use 2ul in a sequencing reaction.
BE PREPARED TO OPTIMIZE!!
(back to top)
Just enter an Evans Fund # on your sequence requistion
form. If you do not have or do not wish to use an Evans account, then
complete a BU or BMC requistion form to submit with your samples. You
will then receive an invoice from BU finance.
(back
to top)
What size tube should I use?
200ul PCR tube should be used to submit samples.
Samples and their duplicates need to be in separate tubes.
(back
to top)
What should the total volume of my submitted sample
be?
Total volume should be 6ul, whether or not you add
your own primer. Please bring sample to final volume using water, NO
buffer.
(back to top)
What primers do you offer?
The Core offers: T7, T3, SP6, M13 forward and reverse,
pGEX3' and pGEX5'. Please refer to the Sequencing page for further information
on these primers. We provide these at NO extra charge.
(back
to top)
When can I expect my results?
The Core runs sequencing plates on Tuesdays and Thursdays.
Results are generally available within 48hours of a run.
(back
to top)
Why did my sample fail/ not work?
- Did you clean the template?
- Did you provide enough DNA (50ng per Kb of template)?
- Does the DNA have a primer site for the primer used?
- Was primer added?
- Is your primer's melting temperature below 55C?
(back to top)
If none of these help, please contact the
Core
Manager with your questions.
Req Form link
Chromas link
