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From 2010.igem.org

-Preparation of pSBIE3 for submission

Digestion

Enzyme DNA is in 0.1ug/uL solution

→ 1uL of each enzyme DNA taken (100ng) for digestion

2.5 uL 10x NEB buffer 4 (best with both EcoRI and PstI)

1uL DNA of enzyme

0.25uL Restriction Enzyme (both)

21.25uL ddH2O

Used “KRIS>DIGEST” program on thermocycler

(*)Made 5uL aliquote (3x) of stock enzyme DNA (initially 50uL of 0.1ug/uL solution)

Enzymes inactivated at 85C for 20 min DNA ligation with pSBIC3 (for 10uL)

E1

E2

E3 add 7uL of inactivated digestion mixture with desired DNA (this is =56ng of DNA)

E4

E5a

E5b