The Stylet

Diversity and Systematics of Acoela and Nemertodermatida

18S F2093/L3449 20071031

Basic information

Procedure

PCR cocktail: 
Component Volume (ul) Mix (ul)
Forward primer  0.5  
Reversed primer 0.5   
Buffer    
MgCl2    
dNTPs    
Taq    
DNA  2  
ddH2O
 22  
Total volume  25
Forward primer(s): 
Thermal cycling (abbreviated): 
95ºC@5min, 2x(95ºC@30sec, 58-54ºC@30sec, 72ºC@30sec), 34x(94ºC@30sec, 52ºC@30sec, 72ºC@60sec), 72ºC@8min
Thermal cycling: 
Step Comment Cycles Time (sec) Temperature (°C)
Initialization For DNA polymerases that need hot-start heat activation      
Denaturation DNA melting producing single-stranded DNA 1
300
 95 
Amplification   2 -  -
Denaturation DNA melting producing single-stranded DNA
 - 30 95
Annealing The primer binds to the template  - 30 58-54 
Extension The DNA polymerase synthesizes new DNA  - 30  72
 Amplification   34    
 Denaturation     30 94
 Annealing     30 52
 Extension     60 72
Final elongation Any remaining single-stranded DNA is extended 1 480  72
Final hold Short-term storage 1 - 8
Reversed primer(s): 
Products and Equipment: 

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Scratchpads developed and conceived by (alphabetical): Ed Baker, Katherine Bouton Alice Heaton Dimitris Koureas, Laurence Livermore, Dave Roberts, Simon Rycroft, Ben Scott, Vince Smith