Noi/NOTES/2012-7-12: Difference between revisions

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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)

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)