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LABORATORY REPORT NO. 09

PRIMER DESIGN WITH PRIMER-BLAST

Correct primers are necessary for successful DNA amplification through PCR
and primer design can be done in multiple ways. It can be painstakingly done
manually or through a multitude of software both online and standalone
applications. Fortunately, the NCBI online BLAST, which you already are familiar
with, has functionality for primer design.

Objectives:
1. To design primer for a specific taxon using NCBI Primer-BLAST (Other
available primer design applications can be used).
2. To appreciate the importance of primer in polymerase chain reaction.

Procedure:

1. Retrieve the sequences you mined in your laboratory report 02.

2. Enter the sequences one after another on the online NCBI Primer-BLAST.
3. Click the button/link named Get Primer at the lower left corner of the page.
4. Document and evaluate results.
5. Consider the following parameters in selecting good primers
GC content between 40% - 60%
The 3’ end ending in G or C
Length of 20 bases (18-30 bases in some cases)
Annealing temperature of 10C (up to at least 50C) between F and R
primers

Results and Discussion

1. Present screenshots of your BLAST results. Interpret results. What primers
are you going to use when you are to amplify extracted DNA from your
group of organisms?
2. Why is primer design necessary?

Generalization
(Make a general statement on your choice of primers.)

Molecular Biology and Diagnostics Laboratory Class; 1st Sem; SY2023-2024 Page 1 of 2
Acknowledgement
(This section is not mandatory. This section is necessary only if there are
individuals, offices or entities that were instrumental in the accomplishment of
this lab report)

Reference/s
Use APA style in listing references.

Appendices
Outline the title of the appended documents starting with Appendix A,
Appendix B and so on. . .

(Attach appendices in correct order)

Molecular Biology and Diagnostics Laboratory Class; 1st Sem; SY2023-2024 Page 2 of 2