I am continuing to design probes for In Situ Hybridization. Currently, I used Ensembl and BLAST to find gene orthologues in the Tiger Salamander (Ambystoma). See Making Primers Part 1 for reference. After I received the primers, I tested them out via a PCR reaction and my cDNA template (generated kindly by our undergrad Minami Tokuyama). 7 out of 10 of the primers seem to work well. This is continuation of the blog Primer Testing and Running a Gel. Note that this article is not peer reviewed and is subject to change.
that I have my gel, I can begin to analyze the results. 3 of the
expected bands do not show up. The other 7 look good, as there are no
strong multiple bands that would indicate non-specific binding. The 100
bp ladder can be read from the bottom up, with the first band in the first well as 100 bp, then next as 200 bp and so on.
that small sequences move more quickly during gel electrophoresis, and
should travel much farther from their starting point (the top of the
gel) towards the bottom. Large sequences are slow and won't move as far.
Therefore, the 5th band from the bottom of 100 bp
DNA ladder, which is much brighter, indicates PCR products that are
roughly 500 base pairs. I can see that seven of my gene products are
between 400 and 600 basepairs.
This is good news to me, as this was the expected outcome.
I consult my spreadsheet to see what the predicted PCR
product sizes are. I had previously determined this information by
entering my primer sequences (forward and reverse) into the Sequence
Manipulation suite: PCR Test, at bioinformatics.org.
can see that in well 2, which is pax6 (exon2, 2nd primer set), should
be 404 base pairs. I can see that in well 4, mef2a (exon1, 2nd primer
set) should be the largest at 546 base pairs. Indeed, it is the largest
sequence and has moved the slowest (from top to bottom, or from the
black negative anode to red positive cathode). Most of the products
should be around roughly 450 bp and this is what my gel reflects.
can see faint Primer Dimer bands which are located under the 100 bp
band (towards the bottom). This doesn't prevent me from using the
primers; however, bands over 100 bp are troublesome as they might
indicate the primers are binding nonspecifically.
After the primers have been tested and deemed correct,
based on predicted PCR product size, reorder the primer with T7.