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= epMotion bead purification testing (continued) = | |||
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | |||
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-11 | |||
* After previous bead purification testing and possible source of contamination, Dinh and I optimize any factor during liquid transfer and mixing step with mock reagents. All optimum parameters and important comments were listed below | |||
== Bead purification optimization notes == | |||
* 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 dispension 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)''' | |||
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 | |||
# 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) |
Revision as of 21:21, 14 July 2012
epMotion bead purification testing (continued)
- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-11
- After previous bead purification testing and possible source of contamination, Dinh and I optimize any factor during liquid transfer and mixing step with mock reagents. All optimum parameters and important comments were listed below
Bead purification optimization notes
- 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 dispension 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) 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
# 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)