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From 2010.igem.org

10-05-2010

PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)

Recipe:

1μL p1003 (approx. 1ng)

2.5μL prA_p1003+

2.5μL prB'_p1003-

5μL 10X PCR buffer

1μL 10uM dNTPs

2μL 50uM MgCl₂

0.5μL Taq polymerase

35.5μL MilliQ H₂O

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Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-.

Program:

1. 5 min-94^oC
2. 45 sec-94^oC
3. 1 min-60^oC
4. 1 min-72^oC
5. Repeat 2 through 4 35 times
6. 5 min-72^oC

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