Wednesday, May 1, 2013

Primer Design Cont.

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 Note that this article is not peer reviewed and is subject to change.

Now 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.

Recall 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

I 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.

I 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.

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