TP17_Picking_primers_for_cloning_and_sequencing_with_the_webserver_Primer3Plus\TP17_Picking_primers_for_cloning_and_sequencing_with_the_webserver_Primer3Plus

1. Using Primer3plus for picking primers for cloning and sequencing

Primer3 is an old, very powerful and widely used program for primer picking. For example, Primer3 was used to pick all primers for the S. cerevisiae genome deletion project. Primer3 can be installed locally, but is also available on-line.

Primer3 is not very user friendly but there is now a web interface for primer3 called primer3plus which is easier to use.

Use this link http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi or google “primer3plus” to find the website for Primer3plus used in this document.

Paste the URA3 sequence (2804 nucleotides) into the main window.

Select Task: Cloning in the upper left corner.

Now, select Task: Cloning in the upper left corner. There are several options, but now we would like to have primers for cloning a specific sequence. Add 1001,804 in the window for Included Regions:

We need to do this to tell Primer3 which sequence we want to clone, in this case the open reading frame of URA3.

The numbers are the start and length of the sequence we want to clone. Click on the “Mark selected region” button

Finally, click on “Pick Primers” in the upper right corner. You should now see an image similar to Figure 2.

You can see that the product size is 804 nt. If you scroll down the page, you will find the sequence with the primers indicated:

As you can see, the Amplified sequence is the ORF of the gene, from the start codon ATG to the stop codon TAA.

Question 1: The SEGUID of the PCR product starts with s9Ykzhe, what are the last four characters of the SEGUID?

1. Using Primer3plus for picking primers for DNA sequencing

Go back to the main window of Primer3plus and choose task: sequencing in the upper left corner. Click Pick Primers. You will see the information below:

If you scroll down, you will see a list of six left (forward) primers.

And six right (reverse) primers:

The purpose of primer picking for sequencing is to choose primers evenly spaced on the sequence, so that every nt on the sequence is covered by at least two sequencing reactions.

The principle is outlined below:

Question 2:

Pick primers for sequencing from the LEU2 locus. This sequence is available in the same folder as this document (LEU2_locus.txt). The size of the first left primer in the list of results should be 21 nucelotides. The SEGUID of the first left primer starts with q7qfNEg, what are the last four characters of the SEGUID?