primer designing and their purpose.

1. Primer Designing
BY- Sanju sah

2. Primer designing:
• Primer
• Purpose????
A. Detection-Gene of interest, Gene expression ( mRNA), Microbial
agents detection, Mutation Detection, Allelic discrimination
B. Cloning
• Single insert cloning
• Multiple insert cloning (cassette construction)
• Site directed mutagenesis

3. Online tools:
•http://www.basic.northwestern.edu/biotools/oligocalc.html
•https://sg.idtdna.com/calc/analyzer
•http://www.ncbi.nlm.nih.gov/
•http://blast.ncbi.nlm.nih.gov/Blast.cgi
•http://reverse-complement.com/
•http://nc2.neb.com/NEBcutter2/

4. Primer Designing
ATCATGGATCCTGTCATACTAGTGTCATAGGTTACTTTCAGCC
Folding
BLAST
GC%= 45/42 Tm= 49.7/66.4 bp= 20/43
Stability of secondary structure=dG= 0.80,Tm= 25.8
Self dimer= -10.76
Heterodimer= -7.79
Restriction sites= BamHI andSpeI
BLAST= √
Homodimer
Heterodimer

7. Primer designing for detection
(for amplification only)

8. Forward Primer
Randomly selected nucleotide stretch for forward primer

27. Reverse Primer Designing

28. Forward Primer
Randomly selected nucleotide stretch for reverse primer

33. Reverse Primer
Rejected Select another region

40. Reverse primer
Forward primer

42. Reverse primer
Select another region Rejected

46. FW-5’GAGTTGGGATCGTTTGTTG 3’
RV-5’ CGATCGTTGGAACATCTTC 3’