AMPure bead purification optimaization (by Dinh & Noi).
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- Labware type:
- The test was performed in 96-well plate (semi-skirt, capacity of the well ~250ul), and all relevance volumes correspond to 96-well plate. However the condition could be applied to other labware like strip tubes or round bottom 96-well plate with miner modification like labware set up (plate or tube) or capacity of the well
- Aspiration step:
- Aspirate beads, 80% EtOH, 20%PEG/2.5M NaCl or H2O/EB buffer (from reservior to well)
- Always aspirate from bottom to avoid different "scan level:level" set up, but make sure that the liquid volume in the reservior is not higher than the tip length
- To aspirate bead, mixing before aspirating is required
- Aspirate spnt or 80% EtOH from well to waste container
- If no aspirate from bottom is noted, used default setting (pipette from top to bottom). Also applied for dispensing from top.
- In case 96-well PCR plate, aspirate from bottom is not recommended for the liquid volume larger than 120ul since the liquid might be forced to splash out and cause contamination.
- In case of the round bottom U-shape or V-shape which have a very large well diameter, aspiration from bottom should ne fine with the volume larger than 120ul, but the threshold need to be measured
- Even the "aspirate volume" is the same vulome as the actual liquid in the well, there is always ~10-20 ul of liquid left over in the well especially using 300ul pipette tip to pipette liquid in large volume. This could be resolved by setting larger "aspirate volume" ~20-40ul.
- Aspirate beads, 80% EtOH, 20%PEG/2.5M NaCl or H2O/EB buffer (from reservior to well)
- Dispensing step:
- After multiple rounds of testing, I would recommend to transfer DNA to the well first and make sure that the liquid is dispensed below the top of the well
- This can be done by unchecked "scan level: level" box before running the application. You could be able to check "scan level: level" box, but make sure that the well to add DNA is empty or has very low level of liquid before.
Pipette reaction to beads plate (liquid type: protein) (PCR products) * Don't check "scan level: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) Discard tips before each aspiration Speed aspiration: 3.0 Speed dispense: 2.0 Delay blow: 0 Speed blow: 4.0 Movement blow: 0 Initial stroke: 100 Prewetting: 0
Pipette beads to beads plate (liquid type: glycerol) Aspirate from bottom Mix before aspirating: 5 cycles, Speed: 7.5, Mix volume: 250 ul, Fixed height (a,d): 1mm, 1mm Mix after dispensing: 10 cycles, Speed: 2.0, Mix volume: 200 ul, Fix height (a,d): 2mm, 2mm Speed aspiration: 3.0 Speed dispense: 2.2 Delay blow: 700 Speed blow: 6.6 Movement blow: 90 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: 0.4 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)
Pipette air to beads plate (liquid type: water) Dispense speed should be very low to prevent liquid spill back to the tip, and this tip is reused to pipette 80% EtOH in some steps Dispense from top, keep tips: do not change tips No mix Speed aspiration: 6.6 Speed dispense: 0.4 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 Volume: 140ul Aspirate from bottom, keep tips: do not change tips No mix Speed aspiration: 3.0 Speed dispense: 2.2 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%) Volume: 180ul 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: 7.5 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) Volume: 50ul Aspirate from bottom, change tips before each aspiration No mix Speed aspiration: 0.4 Speed dispense: 7.5 Delay blow: 0 Speed blow: 17.6 Movement blow: 0 Initial stroke: 100 Prewetting: 0
Resuspend dry beads with 30ul H2O/EB (liquid type: H2O or glycerol or protein if add reaction mix) Volume: 30ul Aspirate from bottom, change tips before each aspiration Mix after dispensing: 20 cycles, Speed: 3.5, Mix volume: 30 ul, Fix height (a,d): 1mm, 1mm Speed aspiration: 3.0 Speed dispense: 3.0 Delay blow: 700 Speed blow: 6.6 Movement blow: 90 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)
Last modified: 2012.07.13 Last modified: 2012.07.18