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

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* Note:
* 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
** 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.
** 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)'''
  '''Pipette beads to beads plate (liquid type: glycerol)'''
   
   

Revision as of 21:36, 14 July 2012

epMotion bead purification testing (continued)

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)

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)