Annealing Oligos

1. Add the following to a 1.5 mL tube at room temperature and mix: 20 uL of each primer, 5 uL sH20, 5 uL T4 Buffer.
2. Set a heat block to 98°C, place tube in heat block, turn off heat block.
3. Allow block to cool down to room temperature, oligos should be annealed.

BP Reaction

1. Add the following components to a 1.5 mL tube at room temperature and mix: attB-PCR product (=10 ng/uL; final amount ~15-150 ng) 1-7 uL , Donor vector (150 ng/uL) 1 uL ,TE buffer, pH 8.0 to 8 uL.
2. Thaw on ice the BP Clonase II enzyme mix for about 2 minutes. Vortex the BP Clonase II enzyme mix briefly twice (2 seconds each time).
3. To each sample (Step 1, above), add 2 uL of BP Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return BP Clonase II enzyme mix to -20°C or -80°C storage.
5. Incluate reactions at 25°C for 1 hour.
6. Add 1 uL of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.
7. Transform 1 uL of each BP reaction into competent cells (see transformation protocol). Plate 20 uL and 100 uL of each on LB plates containing 100 ug/mL kanamycin.

Capped Transcription Reaction Assembly

1. Use mMESSAGE mMACHINE Kit: thaw the frozen reagents [on ice]. Place the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not be frozen at -20°C. Vortex the 10X Reaction Buffer and the 2X NTP/CAP until they are completely in solution. Once thawed, store the ribonucleotides (2X NTP/CAP) on ice, but keep the 10X Reaction Buffer at room temperature while assembling the reaction. All reagents should be microfuged briefly before opening to prevent loss and/or contamination of material that may be present around the rim of the tube.
2. Assemble transcription reaction at room temp. The spermidine in the 10X Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice. Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. The following amounts are for a single 20 uL reaction. Reactions may be scaled up or down if desired.
3. IMPORTANT: the following reaction setup is recommended when the RNA produced will be 300 bp - 5 kb in length. For transcripts longer or shorter than this, see optimization protocols.
4. To 20 uL Nuclease-free Water add: L 2x NTP/CAP, 2 uL 10X Reaction Buffer, 0.1-1 ug Linear template DNA (use 0.1-0.2 ug PCR-product template or 1 ug linearized plasmid template), 2 uL Enzyme Mix.
5. Mix thoroughly. Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube.
6. Incubate at 37C, 1 hr. Typically 80% yield is achieved after a 1 hr incubation. For maximum yield, we recommend a 2 hr incubation. Since SP6 reactions are somewhat slower than T3 and T7 reactions, they especially may benefit from the second hour of incubation. A second hour of incubation is recommended for synthesis of <300 base transcripts and for inefficiently transcribed templates. If the reaction is trace-labelled: after the incubation (before or after TURBO DNase treatment), remove an aliquot of trace-radiolabeled reactions to assess yield by TCA precipitation.
7. (Optional) Add 1 uL TURBO DNase, mix well and incubate 15 min at 37 C. This DNase treatment removes the template DNA. For many applications it may not be necessary because the template DNA will be present at a very low concentration relative to the RNA.

Cell Stock

1. Add 500 uL of overnight LB liquid culture (sequence verified) to a new cryo-vial.
2. Add 500 uL of sterilized 50% glycerol in water to vial.
3. Label tube, store at -80C.

Gate Anneals

Room Temperature Annealing

1. Mix gate and output in a 1.2:1 ratio in a tube.
2. Cover the tube in foil, if you didn't use an opaque/black tube.
3. Leave the tube on a bench top overnight.

Annealing With Heatblock

1. Preheat the heat block to 95°C, add water to the well you will be using.
2. Put a piece of styrofoam (covered with foil) on the heat block. Make sure the temperature is still at 95°C, and doesn't jump
3. Mix gate and output in a 1.2:1 ratio in a tube, put it in the heat block.
4. Cover again with the styrofoam, set the heat block to RT.
5. 5. Wait until the block cools. It will probably not reach RT, but will be at around 30°C after a few hours. Turn the block off completely and wait for a bit (1h).

 N.B. It may be possible to anneal using a lower maximum temperature (say, 80°C), as long as it is higher than the melting temperature of the oligos.

 *Adapted from Qian L, Winfree E. Scaling up digital circuit computation with DNA displacement cascades. , Science. 2011 Jun 3;332(6034):1196-201.

Plate Reader Studies

Kinetic Studies

1. Prepare wells: 100 µL 1X TAE, 12.5 mM Mg2+ buffer; 10 nM 100 µL RNA-ROX (gate); 10 nM 100 µL gate:output complex; 10 nM 100 µL gate:output complex.
2. Measure fluorescence of all wells for 1 minute (5 seconds between measurements).
3. Add 1 µL of the buffer to the buffer and gate wells. Add 1 µl of 1 µM input S6 to the first gate:output well; add 1 µL of 1 µM input S1 to the second gate:output well. Note: Mix well by pipetting into the well, after adding input shake the plate by doing a sort of swirling motion in a plane. If possible, set the plate reader to do this for you for 3 seconds with a 1 second rest time.
4. Measure fluorescence of all wells for 10 minutes (5 seconds between measurements).

 *Adapted from Qian L, Winfree E. Scaling up digital circuit computation with DNA displacement cascades. , Science. 2011 Jun 3;332(6034):1196-201.

In Vitro Transcription

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DNA Transfection

1. Label enough eppendorf tubes for the number of conditions/wells you are transfecting plus one for a master mix of transfection reagent and unsupplemented DMEM. If possible, try to pool conditions/wells, i.e, if doing a small molecule induction ladder.
2. Aliquot 50 uL of unsupplemented DMEM to every tube. If you are pooling n conditions/wells, aliquot 50*n. For your mastermix, aliquot 50* total number of conditions/wells of unsupplemented DMEM.
3. Dilute plasmid DNA in tubes containing 50 (or 50*n) unsupplemented DMEM. Be sure to mix well. For our transfections, we transfect at most 500 ng of plasmid DNA. Be sure that DNA is of high purity and concentrated enough that it can be pipetted in small volumes (from .5 uL- 1 uL). Anything larger will dilute the final concentration.
4. Dilute transfection reagent into unsupplemented DMEM to create the mastermix. We use 1.65 uL Lipofectamine per 500 ng of plasmid DNA per well. So, if transfecting n conditions/wells, dilute 1.65 * n transfection reagent.
5. Add 51.65 uL of master mix per tube and pipette gently to mix. If pooling, mix 51.65 * # of conditions pooled. Let complexes form for 20 minutes in the hood.
6. While complexes are forming, cells must be seeded into a 24 well plate at 100,000 cells in 500 uL. Note. If transfecting in glass bottom well plates, you must pretreat with gelatin (see menu).
7. Trypsinize and count confluent cells. Centrifuge at 1180 rpm for 4 minutes and resuspend in fresh media. Dilute to the 100,000 cells/ 500 uL. Add 500 uL of cells to each well.
8. When complexes are done forming, add dropwise to each well. Swirl the entire plate to mix, and then place into an incubator. Small molecule induction can be done 24 hrs later, and gene expression can be assayed 48 hrs later.

 *Adapted from Invitrogen, Lipofectaime® 2000 Reagent Product Manual.

RNA Transfection

1. Label enough eppendorf tubes for the number of conditions/wells you are transfecting plus one for a master mix of transfection reagent and unsupplemented DMEM. If possible, try to pool conditions/wells, i.e, if doing a small molecule induction ladder.
2. Aliquot 50 uL of unsupplemented DMEM to every tube. If you are pooling n conditions/wells, aliquot 50*n. For your mastermix, aliquot 50* total number of conditions/wells of unsupplemented DMEM.
3. Dilute RNA in tubes containing 50 (or 50*n) unsupplemented DMEM. Be sure to mix well. Be sure that RNA is concentrated enough that it can be pipetted in small volumes (from .5 uL- 1 uL). Anything larger will dilute the final concentration. (insert something here about separate vesicle formation for inputs, G:O etc.

 *Adapted from Invitrogen, Transfecting Stealth™ or siRNA into Mammalian Cells using Lipofectamine™ RNAiMAX.

DNA and RNA Cotransfection

PLACEHOLDER TEXT

Gelatin Pretreatment

This protocol is for use in 24 well, glass bottom, plate format. It can be scaled up or down depending on what size dish you are using.

1. Pipette x uL of .1 (w/v) % sterile, ultrapure gelatin into each well.
2. Let the well plate incubate for at least 20 mins before aspirating off the gelatin.
3. Plates can then be used for transfection, in particular those that will be used for microscopy.

Passaging Cells

Note. This protocol is for culturing cells in 100mm^2 round dishes. For other culture volumes, the amounts of reagents required will be different. Before moving cells from the incubator into the hood,

1. Wipe down hood with 70% EtOH.
2. Pre-warm reagents in a 37 °C water bath. For a full 500 mL bottle of media this can take up to 20 mins to fully warm.
3. Anything moving into the hood, especially from the water bath, must be washed off with 70% EtOH.

Once your hood and reagents are ready,

1. Move cells from incubator to hood.
2. Aspirate off media using a sterilized (autoclaved), glass pastuer pipette, or a disposable aspirating pipette.
3. Optional: Wash cells gently using 2.5 mL PBS, then aspirate.
4. Add 2.5 mL of trypsin to cells. Incubate at most 1 min at 37°C. Work quickly, as over trypsinization will damage the cells.
5. Add 9.5 mL of supplemented media to inactivate trypsin. Pipette to get cells into a single cell suspension.
6. Pipette cells into a conical tube and take note of the final volume (which should be 12mL). This will dictate how much is transferred to a new cell culture dish. If splitting 1:6, then you need 2 mL of cell solution.
7. After transferring cells to new culture dishes, add supplemented media to a final volume of 12-13 mL.
8. Label dishes with Name, Date, Cell type, splitting ratio, and passage number.
9. Place cells into incubator.
10. Destroy unused cells by aspirating into the vacuum trap, or by adding bleach.
11. Wash out of hood with 70% EtOH, close the sash, and turn on UV Light.

Preparing Samples for Flow Cytometry

Note, this protocol is for use with 24 well plates. Trypsin volumes will need to scale according to the plate format you use.

1. Take well plate from the incubator.
2. Aspirate off the supernatant in each well. If cells have not adhered, pipette the supernatant into a conical tube.
3. Add 500 uL of trypsin to each well.
4. Incubate at 37°C for at most a minute.
5. Add 1 mL of supplemented media to stop the trypsinization.
6. Transfer cells to the appropriate conical tube.
7. Centrifuge for 1180 rpm for 4-5 minutes.
8. Apsirate supernatant carefully, not disturbing the cell pellet (it will be rather small).
9. Resuspend in 1 mL of PBS, aiming for a single cell suspension. Any clumps of cells could clog the flow cytometer.
10. Transfer from the conical tube to the 5 mL polystyrene tube for FACS. Any other tube (such as polypropylene) will not work.

Cryopreservation and Cell Thawing

Cryopreservation

This protocol requires the use of Dimethyl Sulfoxide (DMSO), and isopropyl alcohol (IPA). Before using these reagents, you should read a materials safety data sheet (MSDS) for proper handling. Also, for cryopreservation, you ideally want to preserve cells that are at a low passage number (P4 or less).

1. Label as many cryovials as you need (if aiming to freeze down 10*10⁶ cells at 1*10⁶ cells/mL, you need 10 cryovials) with the the passage number of the cells, concentration, initials, date, and cell type. Be sure to also label the caps with something to be able to pick out cryovials easily.
2. Prepare freezing medium by mixing unsupplemented media (DMEM) (50% v/v), FBS (40% v/v) , and DMSO (10% v.v) DMSO is hygroscopic, so you must work quickly when transferring from stocks. Be sure to seal the bottle tightly.
3. Count cells using a hemocytometer (see menu) or an automated cell counter. Dilute cells to a concentration of 1.0*10⁶- 1.5*10⁶ cells/mL.
4. Centrifuge cells at 1180 rpm for 4-5 minutes. Aspirate supernatant and resuspend in freezing media, to a single cell suspension.
5. While cells are in the centrifuge, be sure to check the IPA in the Mr. Frosty freezing chamber. It must be changed every 5th use. Be sure to dispose of IPA properly.
6. Aliquot 1 mL cells to cryovials.
7. Place croyvials in the Mr. Frosty chamber. Freeze at -80°C from 6-24 hrs. Then transfer to either liquid nitrogen tanks, or a -140°C freezer.
8. A few days later, thaw a vial to check cell viability.



Thawing Mammalian Cells

Mamallian cells must be thawed quickly for optimal viability. Since you are removing cells from the -140°C, you need to move quickly and consider extra protection such as a face shield (cryovials can explode).