2ab Assembly

''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