|
Molecular
Biology Protocols |
Last
update April 25, 2005. |
9. Preparing Gel-Purified PCR Products for
Sequencing
1. Check the purity and concentration of your
gel-purified PCR bands by running still another gel, including known DNA
standards for comparison of size and estimation of concentration. Mix 1 μl
of each of the gel-purified PCR products with 9 μl
of 1X TBE and 1 μl of loading dye. Prepare a Low Mass DNA standard in the same
way. Electrophorese
these samples according to the instructions in Protocol 6. From the photograph of the stained gel,
estimate the amount of DNA in each of your samples by comparison with the known
amounts in the standard.
2. For each template, select a primer that is
most likely to hydrogen bond to that template. You may wish to consider
sequencing from each end, using forward primer in one sample and reverse primer
in another. If you have "nested" primers, you may also wish to use
them. Design your sequencing reactions prior to their
assembly, making sure that your primer choice makes sense.
3. The automated DNA sequencer at the Marine DNA
Sequencing Center of MDIBL requires the following for each sequencing reaction:
10 ng template
for every 100 bases of PCR product, up to 80 ng,
dissolved in nuclease-free water
1
microliter oligonucleotide
primer (concentration adjusted to 10 picomoles/microliter
= 10 μmol/liter)
nuclease-free water to a total of 24 microliters
4. Calculate how much template you will need for
a single sequencing reaction and pipet that amount
into a 0.65-ml microcentrifuge tube carefully
labeled as follows:
Your first and last
initial - # (where # represents the number of your specific sample).
5. Add the 1 μl
of primer (at the correct concentration – 10 μmol/liter) plus enough nuclease-free water to
total 24 μl. Be sure to record in your
laboratory notebook which template and primer combination is signified by each
sample number.
6. Submit the samples to Chris Smith at the
Marine DNA Sequence Center of Mount Desert Island
Biological Laboratory, where the samples will be sequenced by a thermal
cycling protocol prior to separation on an ABI Prism 3100 automated sequencer.