HAMILTON MICROLAB® STAR Sequence Controlled Loops

Sequence Controlled Loops Loop continuously until current position marker of controlling sequence goes past end position marker and is therefore set to zero Main use to control loops when you don’t know the length of the controlling sequence in advance i.e Number of positions in sequence used controlled by a user input "How many samples today?" Sequence generated based on worklist

How to set up a Seq controlled loop Select sequences that will be used in asp/dis steps in loop If using fresh tips for each asp/dis step select tip sequence as well Always set reset to never for tip sequence or tip seq will be reset after loop and method will try to pick up tips from positions that have already been used

Why include tip sequence Enables the seq controlled loop to handle the situation where the last loop has less than 8 samples (on 8 channel instrument) On last loop all sequences effectively set to same length as controlling seq If controlling seq only has 4 positions left, tip sequence effectively set to have only 4 positions and 4 tips will be picked up

When not to include tip sequence Do not include tip sequence in list of sequences to iterate over and adjust if keeping same set of tips on for loop (i.e dispensing buffer) Can lead to the incorrect number of channels being used (see advanced user section for more details)

Examples Transfer samples from one plate to another using new tips each time Key Features Include tip seq in seq controlled loop Increment sequence on aspirate and dispense Demo method seq_controlled_loops_demo_fresh_tips

Examples Adding buffer to plate using one set of tips Key Features DO NOT include tip seq in seq controlled loop No sequence increment on asp in order to reuse buffer reservoir Demo method seq_controlled_loops_demo_one_set_tips

HAMILTON MICROLAB® STAR Advanced Users section

How do seq controlled loops work Start of loop Checks condition for running loop is still valid i.e current position marker of controlling seq not zero Sets Max property of each seq included in loop to the Max value of the SHORTEST sequence Max is maximum number of positions available in seq Max = (End – current) +1

How do seq controlled loops work During loop Perform asp/dis step using Max number of positions (if max>number of channels, uses number of channels positions) Sequences incremented as per settings in asp/dis steps (Max is then recalculated at start of next loop based on new current position marker)

Example – transfer samples (fresh tips) Example - What happens at the start of the loop Before loop (Current/end/max) Start of loop 1 (Current/end/max) Source Seq (controlling) 1/20/20 Destination Seq 1/96/96 1/96/20 Tip Seq 1/192/192 1/192/20 8 Channel instrument

How do seq controlled loops work Example - What happens during the loop loop 1 Start (Cur/end) Start (Max) loop 2 loop 2 Start (Max) loop 3 Start (Cur/end) loop 3 Start (Max) loop 4 start (Cur/end) Source Seq (controlling) 1/20 20 9/20 12 17/20 4 0/20 Destination Seq 1/96 9/96 17/96 21/96 Tip Seq 1/192 9/192 17/192 Source, destination and tip seq incremented during asp/dis step in loop

How do seq controlled loops work In loop 3, as Max is set to 4, only 4 tips are picked up and 4 positions used in source and destination sequences At start of loop 4, condition to run loop no longer valid as current position marker of controlling sequence equals Zero. Loop broken and loop 4 does not execute

How do seq controlled loops work What happens if you include tip sequence in seq controlled loop but use same set of tips for loop. Conditions that will cause a pipetting issue Length of pipetting sequences must trigger a second loop Tip sequence must be incremented Number of tips in tip sequence, (End-current)+1, must between one and two times the number of channels used in the pipetting step.

Example – one set tips (tips seq included in seq controled loop) Example – 8 channel instrument loop 1 Start (Cur/end) Start (Max) loop 2 loop 2 Start (Max) Source Seq (i.e buffer) 1/8 8 1 Destination Seq (controlling) 1/24 9/24 Tip Seq 1/9 9/9 As tip seq included in the loop, Max of all sequences set to Max of shortest sequence at start of loop 2 As max is now 1, a single channel will be used to complete seq controlled loop Destination seq and tip seq incremented during asp/dis step in loop

Examples – Using NTRs When using NTR’s and GB NTR DirectUse library, putting tip sequence into loop doesn’t work Example - transfer samples using new tips each time Key Features At start of loop get Max of controlling seq (SeqLib) Create channel pattern based on Max using GB Create_Channel_Pattern library Use gettips_channels step of NTR DirectUse library to pick up tips using channel pattern created

Examples – Using NTRs Example - transfer samples using new tips each time Demo method seq_controlled_loops_NTR_fresh_tips Example – Adding buffer to plate using same set of tips NTRDirectUse gettips_channels step outside of loop and channel pattern based on number of tips required Demo method seq_controlled_loops_NTR_one_set_tips