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Jason Puncheon rifled home a stunning left-foot shot from the edge of the penalty area with ten minutes remaining to send the Coca-Cola League Two team through.
The visiting side were quickly out of the blocks and Jamie Cureton forced Lee Harrison to save after only 30 seconds. Both teams struggled to create attacking opportunities on a difficult playing surface until Cureton broke the deadlock ten minutes before half-time.
Colchester began the second half strongly and had an early chance to double their lead, Kevin McLeod firing just wide from ten yards. Barnet then enjoyed their best spell of the match and equalised in the 62nd minute, the ball ricocheting into the net off Ismail Yakubu after good work from Liam Hatch, a substitute.
The home side continued to create chances, Hatch and Dean Sinclair both going close before Puncheon sealed their passage into the last 32, where they will meet Peterborough United or Plymouth Argyle.
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