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== Bead purification optimization notes (by Dinh & Noi) == * Note: ** The test was performed in 96-well plate (semi-skirt), and all relevance volumes correspond to 96-well plate. However the condition could be applied to other labware like strip tube or round bottom 96-well plate ** Aspiration step, if no aspirate from bottom is noted, used default setting (pipette from top to bottom). Also applied for dispense from top. '''Pipette beads to beads plate (liquid type: glycerol)''' Aspirate from bottom, dispense from top, keep tips Mix before aspirating: 5 cycles, Speed: 7.5, Mix volume: 250 ul, Fixed height (a,d): 1mm, 1mm Speed aspiration: 3.0 Speed dispense: 2.2 Delay blow: 700 Speed blow: 6.6 Movement blow: 90 Initial stroke: 100 Prewetting: 0 '''Pipette reaction to beads plate (liquid type: protein) (PCR products)''' Don't check "check level" option before running the application otherwise the liquid will be dispensed very high from the well and can cause contamination (this is in case that the well already contains 100ul of beads) Aspirate from bottom, discard tips before each aspiration Mix before aspirating: 10 cycles, Speed: 2.0, Mix volume: 180 ul, Fix height (a,d): 1mm, 5mm Speed aspiration: 3.0 Speed dispense: 2.0 Delay blow: 0 Speed blow: 4.0 Movement blow: 0 Initial stroke: 100 Prewetting: 0 '''Pipette solution from beads plate to waste (liquid type: protein)''' Dispense from top, discard tips before each aspiration -> ~20ul larger No mix Speed aspiration: 1.0 Speed dispense: 7.5 Delay blow: 0.0 Speed blow: 4.0 Movement Blow: 0.0 Initial stroke: 100% Prewetting: 0 * Note: have ~20ul liquid left (this is fine in some cases) '''Pipette air to beads plate (liquid type: water)''' Dispense speed should be low Dispense from top, keep tips, do not change tips No mix Speed aspiration: 6.6 Speed dispense: 2.0 Delay blow: 0 Speed blow: 11.0 Movement blow: 0 Initial stroke: 100 Prewetting: 0 '''Pipette ethanol to beads plate (liquid type: Alcohol 75%)''' * Note: edit protocol to add EtOH then mix and dispense column by column Aspirate from bottom, keep tips, do not change tips No mix Speed aspiration: 3.0 Speed dispense: 2.0 Delay blow: 0 Speed blow: 4.0 Movement blow: 0 Initial stroke: 100 Prewetting: 0 '''Pipette ethanol from beads plate to waste (liquid type: Alcohol 75%)''' Aspirate from bottom, Dispense from top, Change tips when command finished Mix before aspirating: 3 cycles, Speed: 2.0, Mix volume: 200ul Fixed height (a,d) : 2mm, 2mm * Note: this because the pipette tips stay too close to the well, and the liquid can not be mixed very well Speed aspiration: 1.0 Speed dispense: 2.2 Delay blow: 0 Speed blow: 4.0 Movement blow: 0 Initial stroke: 100 Prewetting: 0 '''Pipette leftover ethanol from beads plate to waste (liquid type: Alcohol 75%) * Note: use TM_50_(8) Aspirate from bottom, change tips before each aspiration No mix Speed aspiration: 0.4 Speed dispense: 17.6 Delay blow: 0 Speed blow: 17.6 Movement blow: 0 Initial stroke: 100 Prewetting: 0 '''*** Note: need to add''' - Transferring and mixing 20% PEG/2.5M NaCl - Pipette 20%PEG from bead plate to waste - Resuspend 30ul bead with EB buffer or reaction mix # Pipetting air is the trick to balance pipette in and pipette out volume # Go to bottom to aspirate only if the volume in tube is 150 ul or less. # Set mixing to 200ul for first wash so that leftover reagent get mixed & removed. # Add more air to balance the volumes of ethanol removing steps, speed of dispense must be low. # Column by column adding ethanol and then removing with n=8 samples each. # Change to use the correct "plate" for strip tubes. (David's recommendation) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-14
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