Berglund:Typical PCR

Typical PCR reaction

100µl reaction:

76µl water
10µl 10x PCR buffer (http://openwetware.org/wiki/Berglund:PCR_Buffer)
2µl 10mM dNTP (stock mix, A,G, T, C)
3.3µl 10µM forward primer
3.3µl 10µM reverse primer
1µl template (10-100ng)
2µl lab taq

• if the melting temp of your primers is at 57C, then your annealing temp should be two degrees lower (55C), if you see extra bands, you can increase the annealing temp past the melting temp (to about 60°) to increase the stringency, but expect to get less product, so you might have to increase the number of cycles
  
• assume 1000bases/minute for the speed of the lab taq (so if you have a 500bp product- do 40 sec extension, if you have a 2000bp product assume 2 minutes for the extension)
  
• you want the minimum number of cycles to get the right amount of product without mutations
  
  

95° 3 minutes (initial denature step)
95° 40 sec (denature), 55° 40 sec (anneal primers, 72° 1 minute (extension) (repeat this cycle 25-35 times to amplify the product)
72° 2 minutes (final extension)
4° infinity (to cool product until removing it from the thermocycler)