Team:Duke/Notebook/Protocols

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Preparing Chemically Competent Cells

This protocol is for making chemically competent E. coli for transformations. It is derived from the official iGEM protocol, which can be found here

Making CCMB 80 Buffer
- 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
- 80 mM CaCl2.2H2O (11.8 g/L)
- 20 mM MnCl2.4H2O (4.0 g/L)
- 10 mM MgCl2.6H2O (2.0 g/L)
- 10% glycerol (100 ml/L)
- adjust pH DOWN to 6.4 with 0.1M HCl if necessary
Culturing Cells
1. Scrape cells from a colony or frozen stock of the desired strain
2. Inoculate into 5 mL SOC (or LB+Antibiotic for plasmid-containing strains)
3. Grow for ~8 hrs (morning to late afternoon) in 37C shaker
4. Add 5 mL of culture to 250 mL SOC (or LB+Antibiotic) in a shaker flask
  - 250 mL culture will yield approximately 50 chemically competent samples. For smaller batches, we add 1 mL into 50 mL.
5. Grow overnight for ~16 hrs in a shaker at room temperature
Treating cells

Transfer culture into 50 mL centrifuge tubes
Pellet cells at 4500 RPM for 10 mins
Pour off supernatant and resuspend cells in 40 mL CCMB 80 Buffer
Incubate on ice for 20 minutes
Pellet cells at 4500 RPM for 10 mins
Pour off supernatant and resuspend cells in 5 mL CCMB 80 Buffer
Incubate on ice for 20 minutes
Aliquot 750 uL each into pre-chilled microcentrifuge tubes
Store at -80C until use

Note: we never refreeze competent cells once thawed. Any unused cells in an aliquot are discarded.

Transformation

This is our standard heat-shock transformation protocol for E. coli cells. We use it for ligations and other assemblies as well as transformation of intact plasmids (i.e. stocks from the distribution kit).

Thaw chemically competent cells on ice
Add 50 uL cells to 10 uL DNA (plasmid or assembly reaction)
Incubate on ice for 30 minutes
Heat shock for 45 seconds at 42C
Return to ice, incubate for 2 minutes
Transfer cells into 1 mL SOC medium and shake at 37C for 1 hour
Pellet cells at 4500 RPM for 3 minutes and pour off supernatant
Vortex to resuspend and spread on LB+antibiotic agar plates
Incubate overnight at 37C

Ligation

We had some trouble with ligations earlier in the year, and our protocol has undergone changes in an attempt to improve our ligation success. This protocol represents the most recent (and most successful) version of our ligation protocol.

Restriction digestion
- To insert a single part into a desired backbone, digest both the insert and backbone with EcoRI and SpeI
- To insert a new part downstream of an existing part, digest the backbone with PstI and XbaI, and the insert with PstI and SpeI
- Digest four individual tubes of each mini prepped plasmid
- Add 10 uL Cutsmart buffer and 5 uL total enzyme, along with dH20 to a final volume of 100 uL for each mini prep
- Incubate at 37C for 3+ hrs
Gel extraction

Load 200 uL (two digestion tubes) plus 20 uL loading dye into each lane of a 0.8% agarose/TAE gel
Run gel electrophoresis at 100 volts for 18 minutes
Cut desired band from gel
Extract DNA from gel band using Zymoclean gel DNA recovery kit

Antarctic Phosphatase treatment
- Treat only the backbone with phosphatase
- We used Calf Intestinal Phosphatase earlier in the year, but seemed to have better results with Antarctic Phosphatase
- Combine two samples of gel extracted DNA (60 uL) with 10 uL Antarctic Phosphatase buffer, 2.5 uL phosphatase, and dH20 to a final volume of 100 uL
- Incubate for 1-2 hours at 37C
- Clean up sample by running our PCR cleanup protocol using the Qiagen miniprep kit
- Typical yield from minipreps to cleaned, processed DNA is about 10-25%, which is why we start with 4 minipreps to obtain one tube of product.
Ligation
- Combine the following in a 0.5 mL microcentrifuge tube:
  - 100 ng backbone DNA
  - 3x molar excess of insert DNA (ex. 150 ng for an insert half the size of the backbone)
  - 1 uL T4 Ligase buffer
  - 0.5 uL T4 Ligase
  - dH20 to a final volume of 10 uL
- For each reaction, make a backbone-only control and an insert-only control, with an equal volume of dH20 replacing the missing DNA
- Incubate for 1 hour at room temperature (shorter incubation times do not seem to affect efficiency, but this was our default)
- Transform into chemically competent E. coli

PCR preparation of inserts

This protocol was used to isolate PCR products for insertion into BioBrick backbones. It is derived from the Q5 polymerase PCR protocol by New England Biolabs.

Combine the following in individual strip-capped tubes on ice:
- 10 uL 5x Q5 Reaction buffer
- 1 uL dNTPs
- 0.25 uL each primer (100uM)
- 0-1 ng template DNA
- 0.5 uL Q5 DNA polymerase
- dH20 to 50 uL final volume
Run thermocycler protocol:
1. 30 seconds at 98C
2. 35 cycles:
  - 10 seconds at 98C
  - 30 seconds annealing at 2-3C above the lower Tm of the two primers
  - 20-30 seconds per kilobase extension at 72C
3. 10 minutes at 72C
4. hold at 4C
Run 2 uL per tube on a 0.8% agarose/TAE gel for 20 minutes to confirm reaction
Combine tubes and clean up using PCR cleanup protocol

PCR cleanup

We used this protocol to clean up DNA after PCRs, digestions, and other enzyme treatments. It is derived from, and uses materials from, the protocol for the Qiagen spin miniprep kit.

Add 5x volume of Buffer PB to sample, mix and transfer to a spin column.
Spin at 13,000 rpm for 30 seconds. Pour the flow-through back into the column and spin again. Then discard the flow-through.
Add 750 uL of Buffer PE to the column. Spin for 30 seconds, discard the flow-through, then spin again for 1 minute to remove residual buffer.
Transfer the spin column to a labelled microcentrifuge tube. Add 30 uL of Buffer EB, let stand for 3-5 minutes, then spin for 1 minute to elute DNA.

Colony PCR

This is a screening method for identifying transformants after a ligation or other assembly. We used our oligos SB1C3-up and SB1C3-dn with this protocol to screen BioBricks in the standard backbone pSB1C3. We also used our oligos pdCas9-up and pdCas9-dn with this protocol to screen for the addition of crRNA sequences into our pdCas9 plasmid.

Combine the following in individual strip-capped tubes on ice:
- 5 uL Taq buffer
- 1 uL dNTPs
- 0.25 uL each primer (100 uM)
- 0.25 uL Taq polymerase
- dH20 to 50 uL final volume
Prepare a 50 uL SOC tube parallel to each PCR tube
Inoculate each colony from the transformation plate into a PCR tube, then into its parallel SOC tube
Run PCR tubes in thermocycler protocol
Run 3 uL from each PCR tube on a 0.8% agarose/TAE gel to identify successful amplicons
Add SOC copies of successful amplicons to 5 mL LB+antibiotic medium and grow overnight at 37C

Flow Cytometry

We used Flow Cytometry to view the fluorescence of our cells at the individual level. This is the protocol we used when running flow samples.

Culture cells
- Inoculate colonies of desired strains into 5 mL each of LB+antibiotic
- If testing induction with aTc:
  - Inoculate colonies into 50 uL SOC and mix
  - Add 25 uL of SOC mix into medium with aTc/EtOH added
  - Add 25 uL of SOC mix into medium with 100% EtOH added (no aTc)
- Grow to stationary phase (~18 hrs) at 37C
Dilute cultures 1/1000
- Add 5 uL culture to 5 mL equivalent medium
- Grow to mid-log phase for 4.5 hours at 37C
Prepare samples for flow
- Add 20 uL culture to 1 mL PBS (1/50 dilution)
- Place on ice until sample is run
Run samples with the following parameters:
- FSC: log3 scale, 350 Volts
- SSC: log3 scale, 350 Volts
- B1 (GFP/FITC): log5 scale, 350 Volts
- FSC trigger 10.0
- 10,000 events per sample

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