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)
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
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
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
a. Pick colonies of correct growth phenotype into 96 well blocks with
1mL 2YT+correct antibiotics
8. Miniprep if these are intermediate assembly products
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
Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit
MINIPREP Procedure for Plasmid DNA Purification
- Pellet the cells in 96 well block
- spin 5 min at 5500 (use 1 mL of culture if cells were grown in 2YT)
- flick supernatant out into sink
- If cells were grown in LB, pellet another mL of culture before proceeding.
- Do an alkaline lysis
- Use multichannel to add 250uL of Quiagen Buffer P1 into each well.
Resuspend the cells using the vortexer.
- Add 250uL of Quiagen Buffer P2 (a base that denatures everything and
causes cells to lyse). Mix using vortexer Solution should become clearer and blue.
- Add 350uL of Quiagen Buffer N3 buffer (an acid of pH ~5 that causes
cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Mix using the vortexer, make sure all of the blue has disappeared.
- Spin in centrifuge at 5500 for 10 minutes.
- Set up column strips using aluminum holder and 24-well block (to
catch flow through).
- Purify DNA on a column
- Carefully pipette liquid into columns, if you pipette up cell junk,
then re-spin the block at 5500 for 5 min.
- Spin in centrifuge at 2500 for 2 min to pull the liquid through the columns.
- Dump liquid out of the 24-well block (the DNA should be stuck to the
- Wash each column by adding 500 uL of Quiagen Buffer PB.
- Spin in centrifuge at 2500 for 2 min, then flick out the liquid again.
- Wash with 750uL of Quiagen Buffer PE (washes the salts off the resins).
- Spin in centrifuge at 2500 for 2 min, then flick out liquid again.
- Spin in centrifuge at 5500 for 5 min to dry off all water and ethanol.
- Label new 96-well PCR plate and put columns in them.
- Elute DNA by squirting 100uL of water down the middle of the column
(don't let it stick to the sides).
- Spin in centrifuge at 5500 for 3 min. (if the elution volume is very
low, you can spin again)
- Take out columns and cover the plate.
- Clean up - note the A1 buffer is stored at 4degC and all the rest at