Ipswich Town progressed after an entertaining draw was separated by a penalty shoot-out against the U's at Portman Road.
Hayden Mullins opted for a team with five changes to the side that won 4-0 on the road at Sudbury on Friday night, with George, Clampin, Skuse, Jasper and Dobra all missing.
Coming into the side was Turner, Hannant, Nouble, Kennedy and Daniels as the U's looked to progress from the Group Stages of the Papa John's Trophy.
The two sides took no time to find their feet in the final Group Stage game of the Papa John's Trophy, with both Ipswich Town and Colchester United carving out good chances in the opening quarter of an hour.
Freddie Sears worked hard on the right wing to keep the ball in play and win it from Myles Kenlock, driving inside to deliver a low cross to the back post. Frank Nouble was just behind play though, and Kane Vincent-Young was able to put the ball behind and away from danger.
Joe Pigott did have the ball in the net for the hosts inside ten minutes, but fortunately for the U's, his header was offside. The Town forward had strayed into an offside position before nodding back across goal, only to look up and see the linesman holding his flag aloft.
The game was ebbing and flowing from one end to the other and the U's were swiftly on the attack at the other end of the pitch. This time, Sears was looking to control the ball and round Hladky, the the home 'keeper just getting something on the ball to deny the U's from taking the lead.
Pigott was on the attack in front of the U's goal three minutes later, and went closest of all. His powerful strike thumped against the crossbar after a strong hand from Jake Turner to deny the hosts an opening goal.
The U's took the game back to thr he hosts at the other end though, and had their best chance of the game so far. Noah Chilvers delivered a low ball from the right wing towards Luke Hannant who saw his shot blocked away by Burgess just in front of the 'keeper.
There were a couple of further half chances for both sides before the half time whistle blew, but neither side could force the ball home as it remained goalless until the break.
After half time, the U's kept up the good work that they had begun in the first half, with Sears almost finding a route to goal ten minutes in. His effort was spilled, but Chilvers couldn't quite make contact, and the hosts were able to clear.
But the hosts started to apply the pressure, with Jake Turner making plenty of good saves to prevent the home side from scoring.
The involvement of Chay Cooper seemed to reignite the U's, but the youngster missed an open goal in the dying moments which would have handed the U's the win.
The game went to penalties, with both Jake Turner and Vaclav Hladky saving the first spot kicks from Pigott and Sears respectively.
But Ipswich would go on to score their remaining four, with Chaplin, Harper, Celina and Aluko all on target, with Chay Cooper missing the decisive fifth penalty for the U's as the hosts progressed to the next round of the Papa John's Trophy.
Ipswich Town starting XI: Hladky, Harper, Woolfenden, Pigott, Fraser, Barry, Chaplin, Aluko, Vincent-Young (Burns 46'), Burgess, Kenlock (Clements 46').
Unused subs: Holy, Evans, Jackson, Celina, Donacien.
Colchester United starting XI: Turner, Chambers, Wiredu, Hannant, Nouble, Sears, Chilvers, Eastman, Kennedy (Cooper 60'), Tchamadeu (Welch-Hayes 67'), Daniels (Coxe 84').
Unused subs: Gerken, McCoulsky, Thomas, Tovide.