The duplication chance reoccurs when starting a room, even when starting a room by exiting to the main menu and re-entering. This allows you to restart difficult boss fights to get the duplication you want (i.e. a duplicated Psy Fly for Delirium fight). Of course the fight will restart if you do this, but if you do this immediately upon entering the room there is no cost.
Synergies[ | ]
Golden Trinket/Mom's Box/The Twins: Each effect/trinket copy stacks granting an additional 50% chance to trigger the effect again per stack. This results in another familiar doubling. This can be the same familiar that was doubled originally. The duplication chance of three or more stacks can go above 100% allowing for even more duplicate familiars or Brother Bobbys/Sister Maggys to be created.
Sacrificial Altar: Both the duplicated familiar (if one is available) and Brother Bobby/Sister Maggy (if not available) can be sacrificed for an item reward. This does not remove the trinket, and fully retains its effect.
In the case of Shade, a copy of the familiar will spawn even if Shade was already absorbed by Isaac.
Pickup-generating familiars: If they remain duplicated for enough consecutive rooms, they will give a pickup as normal. The following familiars have to be duplicated for the following number of consecutive room clears to give pickups:
Sack of Pennies: 1 room (will give a coin every time it’s duplicated)
Because Lil Chest and Charged Baby’s chances to spawn pickups are independent from the total number of rooms cleared with them, they have an extra chance to pay out in any room they’re duplicated in.
1up!: Despite the fact that it can be duplicated, Isaac will not keep the 1up if he dies while it's duplicated.
GB Bug: The familiar can be duplicated even if it hasn’t respawned; by constantly moving to new rooms, GB Bug can constantly respawn, allowing pickups to be rerolled indefinitely.
Guillotine: The head can be duplicated, and will give an extra damage and tears up if it is. The extra head will be unable to fire tears.
Hallowed Ground: Duplicate poops will also drop when Isaac takes damage. The bonuses from standing in the aura of multiple white poops does not stack.
Lost Soul / Star of Bethlehem: The trinket can try to make a duplicate of the familiar, but it will always fail. This reduces the odds of the trinket giving copies of other familiars or prevents it from giving Brother Bobby or Sister Maggy if Isaac has no other familiars.
Mystery Egg: The friendly the duplicated egg spawns depends on how many consecutive rooms cleared the duplicate persisted for.
Strawman: Keeper cannot be duplicated, as he is considered a character, not a familiar.
Succubus: The damage multiplier from standing in the aura of multiple Succubi multiplicatively stacks.
YO LISTEN!: An extra +1 luck will be given for every duplicate.