"Fishing" Out Microsatellites
Primer Design Practice
To practice designing primers, print out this page but do not close it to preserve the hot links below.
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Click on the National Center for Biotechnology Information web site. The link can be accessed by clicking on the name above.
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In the block following "Search Nucleotide for," enter: microsatellite pme2
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Click on the pme2 identification number, "AF362445"
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You should have a listing of information on the sequence of the pme2 microsatellite. Go to the bottom of the page and click at the beginning of the base sequence and drag the cursor to the end of the sequence. Click on Edit, Copy.
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Open Microsoft Word and Paste the sequence onto a new document in Word. Edit the sequence by deleting the // at the end of the sequence and edit out all numbers and spaces so the base sequence is continuous throughout.
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Highlight the continuous sequence and click Edit, Copy.
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Click on the Primer3 web site. The link can be accessed by clicking here or on the Primer Design page.
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Paste the continuous sequence into the large box at the top of the Primer 3 web page.
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Scroll down and enter the following in the appropriate places. The first column is the tag you should see on the page. The second column below is the information to put into the boxes.
| Seq. ID | pme2 | |
| Targets | 156,36 | |
| (This indicates that the target sequence you want to include starts at base 156 and is 36 bases long. This is the series of CA repeats in the microsatellite sequence you are using.) | ||
| Product Size Max | 500 | |
| Primer GC% Opt | 50 | |
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Scroll down until you see the next Pick Primers tab and click on it.
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Finally, to test out your primers further, print out the Primer3 Output page and enter the primer sequences in the program Amplify (see link on Primer Design page) and follow the instructions given there.
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