WIP - Add colony picking protocols

Plasmids extraction/README

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+Manual protocol for Miniprep for plasmid extraction in 96 well plate format
+
+Reagents needed:
+-LB + Antibiotics
+-Buffer P1 (Quiagen)     50 mM Tris-HCl pH 8.0; 10 mM EDTA; 100 μg/ml RNaseA  (33.6mL for one plate)
+-Buffer P2 (Quiagen)     200 mM NaOH; 1% SDS                                  (33.6mL for one plate)
+-Buffer P3 (Quiagen)     3.0 M potassium acetate pH 5.5                       (33.6mL for one plate)
+-100% Isopropanol
+-70% Ethanol
+(Buffer composition of Quiagen https://openwetware.org/wiki/Qiagen_Buffers)
+
+Protocol:
+1- Colonies are inoculated in 1000uL media LB + Antibiotics, incubation overnight 37°C in a sealed 96 well with shaking
+2- Harvest the bacterial cells by centrifugation at 6000g for 15min at 4°C. Supernatant is discarded by inverted the plate upside down.
+3- Resolubize the cell pellet by adding 350uL of buffer P1 and do up and down with the pipette. (If needed, vortex vigorously to help resuspend the bacteria)
+4- Transfer with 350uL of buffer P2. Mix by inverted sealed plate 4-6 times.
+5- Incubates plates at room temperature for 5min.
+6- Transfer with 350uL of buffer P3. Mix by inverted sealed plate 4-6 times.
+7- Pellet the debris by centrifuging the plate at 6000g for 45min at 4°C
+8- Transfer 900uL of supernatant into the new plate. (be careful to avoid touching the precipitate. If it is the case, repeat step 7)
+9- Precipitate DNA by adding 600uL of isopropanol in each well
+10- Mix by vortexing and centrifuge plates at 6000g for 45min at 4°C. Discard supernatant 
+11- Wash DNA pellet with 1000uL of 70% ethanol, and centrifuge at 6000g for 15min
+12- Repeat step 11
+13- Invert the plate on towel, then let the ethanol evaporate by air-drying the plate for 15-30min
+14- Redissolve the DNA in 200uL sterile water 

Plasmids extraction/miniprep_96_well

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+#!/usr/bin/env python
+# coding: utf-8
+
+"""Opentrons python protocol adapted from """
+
+from opentrons import protocol_api
+
+metadata = {
+    "protocolName": "Screening from inoculated culture",
+    "author": "YHK",
+    "description": "Miniprep from culture in 96 deep-well plate",
+    'apiLevel': '2.11'}
+
+def run(protocol: protocol_api.ProtocolContext):
+    pellet = protocol.load_labware('nest_96_wellplate_2ml_deep', 2)
+    supernatant = protocol.load_labware('nest_96_wellplate_2ml_deep', 1
+    )
+    buffer_mini = protocol.load_labware('nest_12_reservoir_15ml', 4)
+    isopropanol = protocol.load_labware('brand_1_reservoir_8000ul', 7)
+    ethanol = protocol.load_labware('brand_1_reservoir_8000ul', 8)
+
+
+    tiprack = protocol.load_labware('opentrons_96_tiprack_300ul', 6)
+    tiprack2 = protocol.load_labware('opentrons_96_tiprack_300ul', 5)
+    tiprack3 = protocol.load_labware('opentrons_96_tiprack_300ul', 3)
+
+    p300 = protocol.load_instrument('p300_multi_gen2', 'right', tip_racks=[tiprack, tiprack2, tiprack3])
+
+    # loop to transfert solution from buffer to pellet with mixing
+
+    for i in range(3):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[0]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest.top(), new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 2.5)
+        p300.return_tip()
+
+    for i in range(3,6):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[1]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 2.5)
+        p300.return_tip()   
+
+    for i in range(6,9):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[2]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 2.5)
+        p300.return_tip() 
+
+    for i in range(9,12):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[3]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 2.5)
+        p300.return_tip()   
+
+
+    # loop for P2
+
+    for i in range(3):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[4]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 1.5)
+        p300.return_tip()
+
+    for i in range(3,6):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[5]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 1.5)
+        p300.return_tip()   
+
+    for i in range(6,9):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[6]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 1.5)
+        p300.return_tip() 
+
+    for i in range(9,12):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[7]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2), rate= 1.5)
+        p300.return_tip()  
+
+    # Pause for 4min
+    protocol.delay(minutes=4)
+
+
+    # inactivation of the lysis by adding 350uL P3
+
+    for i in range(3):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[8]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2))
+        p300.return_tip()
+
+    for i in range(3,6):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[9]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2))
+        p300.return_tip()   
+
+    for i in range(6,9):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[10]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2))
+        p300.return_tip() 
+
+    for i in range(9,12):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[11]
+        dest = pellet.columns()[i]
+        p300.transfer(350, source, dest, new_tip = 'never')
+        p300.mix(5, 200, pellet.wells()[8*i].bottom(2))
+        p300.return_tip()  
+
+    # transfert of 900ul of buffer to a new plate and put new tips box
+
+    protocol.pause('change tips box')
+    p300.reset_tipracks()
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = pellet.wells()[8*i]
+        dest = supernatant.columns()[i]
+        p300.transfer(850, source.bottom(2), dest, new_tip = 'never')
+        p300.return_tip() 
+
+    # transfert of 600ul of isopropanol to precipitate
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = isopropanol.wells()[0]
+        dest = supernatant.columns()[i]
+        p300.transfer(600, source, dest, new_tip='never')
+        p300.return_tip()
+
+    # pause 
+    protocol.pause('vortex and centrifugation')
+
+    # Wash DNA pellet with 1000uL of 70% ethanol
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = ethanol.wells()[0]
+        dest = supernatant.columns()[i]
+        p300.transfer (1000, source, dest, new_tip= 'never')
+        p300.mix(5, 200, supernatant.wells()[8*i])
+        p300.return_tip() 
+
+    # pause to centrifuge 6000g for 15min
+
+    protocol.pause('centrifugation 6000g for 15min')
+
+    # Wash DNA pellet with 1000uL of 70% ethanol, second
+
+    protocol.pause('change tips box')
+    p300.reset_tipracks()
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = ethanol.wells()[0]
+        dest = supernatant.columns()[i]
+        p300.transfer (1000, source, dest, new_tip= 'never')
+        p300.mix(5, 200, supernatant.wells()[8*i])
+        p300.return_tip() 
+
+    # pause to centrifuge 6000g for 15min
+
+    protocol.pause('centrifugation 6000g for 15min')
+
+    # Redissolve the DNA in 200uL sterile water 
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = buffer_mini.wells()[9]
+        dest = supernatant.wells()[8*i]
+        p300.transfer (1000, source, dest, new_tip= 'never')
+        p300.mix(10, 100, supernatant.wells()[8*i], rate =2.0)
+        p300.return_tip()

Plasmids extraction/principle_and_protocol

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+Manual protocol for Miniprep for plasmid extraction in 96 well plate format
+
+Reagents needed:
+-LB + Antibiotics
+-Buffer P1 (Quiagen)     50 mM Tris-HCl pH 8.0; 10 mM EDTA; 100 μg/ml RNaseA  (33.6mL for one plate)
+-Buffer P2 (Quiagen)     200 mM NaOH; 1% SDS                                  (33.6mL for one plate)
+-Buffer P3 (Quiagen)     3.0 M potassium acetate pH 5.5                       (33.6mL for one plate)
+-100% Isopropanol                                                             (57,3mL for one plate)
+-70% Ethanol                                                                  (192mL for one plate)
+(Buffer composition of Quiagen https://openwetware.org/wiki/Qiagen_Buffers)
+
+Protocol:
+1- Colonies are inoculated in 1000uL media LB + Antibiotics, incubation overnight 37°C in a sealed 96 well with shaking
+2- Harvest the bacterial cells by centrifugation at 6000g for 15min at 4°C. Supernatant is discarded by inverted the plate upside down.
+3- Resolubize the cell pellet by adding 350uL of buffer P1 and do up and down with the pipette. (If needed, vortex vigorously to help resuspend the bacteria)
+4- Transfer with 350uL of buffer P2. Mix by inverted sealed plate 4-6 times.
+5- Incubates plates at room temperature for 5min.
+6- Transfer with 350uL of buffer P3. Mix by inverted sealed plate 4-6 times.
+7- Pellet the debris by centrifuging the plate at 6000g for 45min at 4°C
+8- Transfer 900uL of supernatant into the new plate. (be careful to avoid touching the precipitate. If it is the case, repeat step 7)
+9- Precipitate DNA by adding 600uL of isopropanol in each well
+10- Mix by vortexing and centrifuge plates at 6000g for 45min at 4°C. Discard supernatant 
+11- Wash DNA pellet with 1000uL of 70% ethanol, and centrifuge at 6000g for 15min
+12- Repeat step 11
+13- Invert the plate on towel, then let the ethanol evaporate by air-drying the plate for 15-30min
+14- Redissolve the DNA in 200uL sterile water 

paper_blot/paperblot.py

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+#!/usr/bin/env python
+# coding: utf-8
+
+"""Opentrons python protocol adapted from """
+
+from opentrons import protocol_api
+
+metadata = {
+    "protocolName": "Paper Blot",
+    "author": "YHK",
+    "description": "Drying liquid on whatman paper/Membrane",
+    'apiLevel': '2.11'}
+
+def run(protocol: protocol_api.ProtocolContext):
+    samples = protocol.load_labware('nest_96_wellplate_2ml_deep', 2)
+    membrane = protocol.load_labware('nest_96_wellplate_2ml_deep', 1)
+    tiprack = protocol.load_labware('opentrons_96_tiprack_300ul', 6)
+
+
+    p300 = protocol.load_instrument('p300_multi_gen2', 'right', tip_racks=[tiprack])
+
+    # loop to transfert solution from buffer to pellet with mixing
+
+    for i in range(12):
+        p300.pick_up_tip()
+        source = samples.columns()[i]
+        dest = membrane.wells()[8*i]
+        p300.transfer(2, source, dest.top(), new_tip = 'never')
+        p300.return_tip()
+
+

paper_blot/test_paper_blot.py

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+#!/usr/bin/env python
+# coding: utf-8
+
+"""Opentrons python protocol adapted from """
+
+from opentrons import protocol_api
+
+metadata = {
+    "protocolName": "Paper Blot",
+    "author": "YHK",
+    "description": "Drying liquid on whatman paper/Membrane",
+    'apiLevel': '2.11'}
+
+def run(protocol: protocol_api.ProtocolContext):
+    samples = protocol.load_labware('nest_12_reservoir_15ml', 2)
+    membrane = protocol.load_labware('127x85_agar_plate1', 1)
+    tiprack = protocol.load_labware('opentrons_96_tiprack_300ul', 6)
+
+
+    p20 = protocol.load_instrument('p20_multi_gen2', 'right', tip_racks=[tiprack])
+
+    # loop to transfert solution from buffer to pellet with mixing
+
+    for i in range(12):
+        p20.pick_up_tip()
+        source = samples.wells()[1]
+        dest = membrane.wells()[8*i]
+        p20.aspirate(1.5, source) 
+        p20.touch_tip()
+        p20.dispense(1.5, dest.top())
+        p20.return_tip()

picking_robot/README

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+PLease readme

picking_robot/principle_and_protocol

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+Screening on plate from liquid culture
+
+The principle of this protocol is to transfer liquid culture of transformants
+on agar plate to screen different condition. In that protocol, 4 96_well plate
+culture of unique transformant are transfer on 3 square agar plate.
+
+Important: the custom plate definition must be on '384Standard' format and
+not on 'irregular' format. Otherwise, there would be error with accessing 
+the B row with a 8 channel pipette