2ab Assembly

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(New page: ''Note: You can download this excel file for 2AB Robot Assembly Media:2AB_RobotAssembly.xls. It contains buffer calculators.''<br> An example worklist: [[Media:MediatorProjectWorklist....)
Current revision (16:39, 7 June 2010) (view source)
 
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1. '''Make a "dilution plate"''' (96 well)
1. '''Make a "dilution plate"''' (96 well)
-
  The robot can pipette a minimum volume of 3uL accurately. Miniprepped
+
  The robot can pipette a minimum volume of 3uL accurately. Miniprepped DNA will need to be diluted for the 2AB Robot reactions.
-
DNA will need to be diluted for the 2AB Robot reactions.
+
  a. Calculate the total volume of DNA needed for the assembly reaction
  a. Calculate the total volume of DNA needed for the assembly reaction
-
     i. 3uL per reaction + 5uL extra (you need the extra volume in the
+
     i. 3uL per reaction + 5uL extra (you need the extra volume in the well to ensure the robot pipettes accurately)  
-
well to ensure the robot pipettes accurately)
+
  b. If you miniprepped your DNA with the Macherey Nagel strips, dilute the DNA two fold into NEB2  ie- equal volumes of DNA and 2X NEB2
-
  b. If you miniprepped your DNA with the Macherey Nagel strips, dilute
+
-
the DNA two fold into NEB2  ie- equal volumes of DNA and 2X NEB2
+
     i. Make sure each well has the volume of DNA calculated in (a)
     i. Make sure each well has the volume of DNA calculated in (a)
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     i. '''Robot Protocol:''' 032909-2ab-BamBglCocktail
     i. '''Robot Protocol:''' 032909-2ab-BamBglCocktail
     ii. '''Command File:''' 2ab-LR.csv [[Media:EXAMPLE_2AB-LR.xls]]
     ii. '''Command File:''' 2ab-LR.csv [[Media:EXAMPLE_2AB-LR.xls]]
-
     iii. Always make sure the robot has enough tips and that you have
+
     iii. Always make sure the robot has enough tips and that you have changed the .csv file to the correct one for your assembly
-
changed the .csv file to the correct one for your assembly
+
  b. Cherry pick out lefty and righty plasmids (3uL of each)
  b. Cherry pick out lefty and righty plasmids (3uL of each)
     i. '''Robot Protocol:''' 032909-2abPartTransfer
     i. '''Robot Protocol:''' 032909-2abPartTransfer
     ii. '''Command File:''' 2ab-LR.csv [[Media:EXAMPLE_2AB-LR.xls]]
     ii. '''Command File:''' 2ab-LR.csv [[Media:EXAMPLE_2AB-LR.xls]]
  c. Cover the reaction plate with foil
  c. Cover the reaction plate with foil
-
  d. Run "123" on black thermocycler
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  d. Run "123" on black thermocycler (incubate at 37 for 1hr, heat kill 65 for 20min)
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  (incubate at 37 for 1hr, heat kill 65 for 20min)
+
3.''' Ligate'''
3.''' Ligate'''
  a. Distribute 4uL Ligase Cocktail to each well
  a. Distribute 4uL Ligase Cocktail to each well
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  b. Add 30 uL of competent cell solution to each well
  b. Add 30 uL of competent cell solution to each well
     i. If using the 1.5mL aliquots of pir-Righty or pir-Lefty add 200uL of KCM
     i. If using the 1.5mL aliquots of pir-Righty or pir-Lefty add 200uL of KCM
-
     ii. Note that if this is the last step in your assembly you may
+
     ii. Note that if this is the last step in your assembly you may want to transform into an assay cell type
-
want to transform into an assay cell type
+
  c. Incubate on ice for 10 min
  c. Incubate on ice for 10 min
  d. Heat shock 90 sec at 42C
  d. Heat shock 90 sec at 42C
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  d. Plate 40uL on LB agar strips
  d. Plate 40uL on LB agar strips
  e. Dry on bench top
  e. Dry on bench top
-
  f. Stack plates, cover the top one with airpore, invert, incubate
+
  f. Stack plates, cover the top one with airpore, invert, incubate overnight at 37C
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overnight at 37C
+
6. '''Pick colonies'''
6. '''Pick colonies'''
  a. Use sterile tooth picks to pick 3 colonies/strip
  a. Use sterile tooth picks to pick 3 colonies/strip
  b. Spot check on AKC and dual antibiotic plates
  b. Spot check on AKC and dual antibiotic plates
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     i. you need to spot them on both types of plates to check for
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     i. you need to spot them on both types of plates to check for co-tranformation and to have colonies for the next day
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co-tranformation and to have colonies for the next day
+
  c. Incubate plates at 37C overnight
  c. Incubate plates at 37C overnight
7. '''Innoculate'''
7. '''Innoculate'''
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  a. Pick colonies of correct growth phenotype into 96 well blocks with
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  a. Pick colonies of correct growth phenotype into 96 well blocks with 1mL 2YT+correct antibiotics<br>
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1mL 2YT+correct antibiotics<br>
+
8.''' Miniprep''' if these are intermediate assembly products
8.''' Miniprep''' if these are intermediate assembly products
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9. '''Screen the assembly products'''
9. '''Screen the assembly products'''
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  a. If you have miniprepped DNA (ie- you are screening intermediate
+
  a. If you have miniprepped DNA (ie- you are screening intermediate assembly products), run a [[ArkinLab_ALD_RestrictionMapping | Restriction Digest]]
-
assembly products), run a [[ArkinLab_ALD_RestrictionMapping |
+
-
Restriction Digest]]
+
  -or-
  -or-
  b. If you do not have miniprepped DNA (ie- your finished products
  b. If you do not have miniprepped DNA (ie- your finished products
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10. '''Start again''' at (1) until goal composite parts are made
10. '''Start again''' at (1) until goal composite parts are made
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===Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit===
 
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'''MINIPREP Procedure for Plasmid DNA Purification'''
 
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*Pellet the cells in 96 well block
 
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#spin 5 min at 5500 (use 1 mL of culture if cells were grown in 2YT)
 
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#flick supernatant out into sink
 
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#If cells were grown in LB, pellet another mL of culture before proceeding.
 
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*Do an alkaline lysis
 
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#Use multichannel to add 250uL of Quiagen Buffer P1 into each well.
 
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Resuspend the cells using the vortexer.
 
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#Add 250uL of Quiagen Buffer P2 (a base that denatures everything and
 
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causes cells to lyse). Mix using vortexer Solution should become
 
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clearer and blue.
 
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#Add 350uL of Quiagen Buffer N3 buffer (an acid of pH ~5 that causes
 
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cell junk - including protein and chromosomal DNA - to precipitate,
 
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and leaves plasmids and other small molecules in solution). Mix using
 
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the vortexer, make sure all of the blue has disappeared.
 
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#Spin in centrifuge at 5500 for 10 minutes.
 
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#Set up column strips using aluminum holder and 24-well block (to
 
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catch flow through).
 
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*Purify DNA on a column
 
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#Carefully pipette liquid into columns, if you pipette up cell junk,
 
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then re-spin the block at 5500 for 5 min.
 
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#Spin in centrifuge at 2500 for 2 min to pull the liquid through the columns.
 
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#Dump liquid out of the 24-well block (the DNA should be stuck to the
 
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white resin)
 
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#Wash each column by adding 500 uL of Quiagen Buffer PB.
 
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#Spin in centrifuge at 2500 for 2 min, then flick out the liquid again.
 
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#Wash with 750uL of Quiagen Buffer PE (washes the salts off the resins).
 
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#Spin in centrifuge at 2500 for 2 min, then flick out liquid again.
 
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#Spin in centrifuge at 5500 for 5 min to dry off all water and ethanol.
 
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#Label new 96-well PCR plate and put columns in them.
 
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#Elute DNA by squirting 100uL of water down the middle of the column
 
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(don't let it stick to the sides).
 
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#Spin in centrifuge at 5500 for 3 min. (if the elution volume is very
 
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low, you can spin again)
 
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#Take out columns and cover the plate.
 
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#Clean up - note the A1 buffer is stored at 4degC and all the rest at
 
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room temperature.
 

Current revision

Note: You can download this excel file for 2AB Robot Assembly Media:2AB_RobotAssembly.xls. It contains buffer calculators.
An example worklist: Media:MediatorProjectWorklist.xls

1. Make a "dilution plate" (96 well)

The robot can pipette a minimum volume of 3uL accurately. Miniprepped DNA will need to be diluted for the 2AB Robot reactions.
a. Calculate the total volume of DNA needed for the assembly reaction
   i. 3uL per reaction + 5uL extra (you need the extra volume in the well to ensure the robot pipettes accurately) 
b. If you miniprepped your DNA with the Macherey Nagel strips, dilute the DNA two fold into NEB2  ie- equal volumes of DNA and 2X NEB2
   i. Make sure each well has the volume of DNA calculated in (a)

2. Digest (make a "reaction plate")

a. Distribute 10uL BamBgl Cocktail to each well
     BamBgl Cocktail
         1    Neb2
         0.5  BamHI
         0.5  BglII
         0.5  XhoI
         7.5  water
      10uL total
   i. Robot Protocol: 032909-2ab-BamBglCocktail
   ii. Command File: 2ab-LR.csv Media:EXAMPLE_2AB-LR.xls
   iii. Always make sure the robot has enough tips and that you have changed the .csv file to the correct one for your assembly
b. Cherry pick out lefty and righty plasmids (3uL of each)
   i. Robot Protocol: 032909-2abPartTransfer
   ii. Command File: 2ab-LR.csv Media:EXAMPLE_2AB-LR.xls
c. Cover the reaction plate with foil
d. Run "123" on black thermocycler (incubate at 37 for 1hr, heat kill 65 for 20min)

3. Ligate

a. Distribute 4uL Ligase Cocktail to each well
     Ligase Cocktail  
         0.4  Neb2
         2    10mM ATP
         0.5  Ligase
         1.1  water
      4uL total
   i. Robot Protocol: 032909-2ab-Ligase
   ii. Command File: 2ab-LR.csv Media:EXAMPLE_2AB-LR.xls
b. Incubate room temp 30 min

4. Transform

a. Put reaction plate on ice
   i. Use silver cold block
b. Add 30 uL of competent cell solution to each well
   i. If using the 1.5mL aliquots of pir-Righty or pir-Lefty add 200uL of KCM
   ii. Note that if this is the last step in your assembly you may want to transform into an assay cell type
c. Incubate on ice for 10 min
d. Heat shock 90 sec at 42C
   i. Use black thermocycler
e. Ice 1 minute
f. Add 100uL of plain (no antibiotics) LB or 2YT
g. Resuce at 37C for 45 minutes

5. Plate

a. Spin down cells at 5300 for 3 min
b. Remove 80uL of supernatant
c. VERY GENTLY resuspend cell pellet in the remaining liquid
d. Plate 40uL on LB agar strips
e. Dry on bench top
f. Stack plates, cover the top one with airpore, invert, incubate overnight at 37C

6. Pick colonies

a. Use sterile tooth picks to pick 3 colonies/strip
b. Spot check on AKC and dual antibiotic plates
   i. you need to spot them on both types of plates to check for co-tranformation and to have colonies for the next day
c. Incubate plates at 37C overnight

7. Innoculate

a. Pick colonies of correct growth phenotype into 96 well blocks with 1mL 2YT+correct antibiotics

8. Miniprep if these are intermediate assembly products

 8-Strip Machery Nagel Miniprep

9. Screen the assembly products

a. If you have miniprepped DNA (ie- you are screening intermediate assembly products), run a  Restriction Digest
-or-
b. If you do not have miniprepped DNA (ie- your finished products

were transformed directly into an assay cell line), run Colony PCR

10. Start again at (1) until goal composite parts are made

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