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From 2010.igem.org

Digestion

1. Digestion of PCR products and pSB1A2
   1. PyodA-mAAA : EcoRI and SpeI
   2. PzntA-RFP : XbaI and PstI
   3. pSB1A3 : EcoRI and PstI

Reagent	Volume (μL)
DNA (about 30ng/μL)	30
10x NEB buffer 2	5
BSA (10mg/mL)	0.5
Appropriate 1st and 2nd restriction enzymes	2 (each 1)
D.W.	12.5 / total = 50
Completely digestion at 37˚C for 2 hours (at least) and stop at 80˚C for 20min

1. Confirmation of digested products by agarose gel electrophoresis (Figure 4)
2. Quantification of DNA concentration by NanoDrop