Experiments

Protocols

PCR (Polymerase Chain Reaction)

Reactants

10x Reaction Mix	5 uL
For primer (10 uM)	1 uL
Rev primer (10 uM)	1 uL
Template	1 uL
Taq Polymerase	1 uL
MgCl2 (25 mM)	1 uL
DMSO	1 uL
NF H2o	41 uL
Total	50 uL

Protocol

On ice, add all reagents to PCR tube, Taq polymerase last.
Run thermocycler program:

95 C for 5 min (2 min if NOT using Hotstart)

> 10 cycles:
95 C for 20 s
0.3 C/s to 50 C
72 C for # s (extension = 1 min/kb)

> 15 cycles:
95 C for 20 s
55 C for 20 s
72 C for 1 min

72 C for 10 min
4 C forever

PCR Clean Up

Reagents and protocol from Zymo DNA Clean & Concentrator Kit

Reagents

DNA Binding Buffer	Refer to table below.
DNA Wash Buffer	Refer to table below.
NF H2O	Volume dependent on objective.

Protocol:all centrifugation steps should be performed between 10,000 - 16,000g.

On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.

Application	DNA Binding Buffer: Sample	Example
Genomic DNA (> 2 kb)	2 : 1	200 uL : 100 uL
PCR Product, DNA Fragment	5 : 1	500 uL : 100 uL

Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.
Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x.
Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.
Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.

Gels & Gel Electrophoresis

Reagents:

TAE Buffer	~ 50 mL per gel, more necessary for electrophoresis chamber.
NF Agarose (generally Seakem LE)	Grams of agarose : mL buffer = percentage of gel
Ethidium Bromide	~ 1 drop

**Don’t forget ladder.
Protocol (gel):

Mix the TAE buffer and agarose once measured in microwavable flask. Stir with stir bar in flask and magnetic stirrer.
Afterwards, microwave for 1-3 mins until agarose is completely dissolved.
Let the flask cool until it is cool enough to carry with the orange heat “gloves.”
Add ethidium bromide and let gel solidify (10 - 20 min). Remember combs.
Usually, use the gel for gel electrophoresis right away, but if needed, the gel can be stored in a container filled with TAE buffer and 1-2 drops of ethidium bromide.

Protocol (gel electrophoresis):

Add reagents together (always at least ladder and sample).

This can occur in PCR tubes or parafilm.

Place the gel in the gel tray in the gel electrophoresis chamber and fill with TAE buffer to max line.
- Since DNA is negatively charged, the wells should be near the positive end (black) and DNA will “run to red.”
Put the ladder and sample(s) in the wells with the ladder in Well 1, etc. Close the chamber properly, and run the gel electrophoresis chamber at 100 - 130 V.
Wait for about 30 minutes before stopping the gel electrophoresis chamber and removing the gel tray.
Place the gel in the Gel Imager (UV Light) next to the computer that will show the image. Run the program “Ethidium Bromide” to see DNA bands.
- Make sure to position the gel in the center.
- Remember: smaller fragments are farther away from the wells.
Clean up area, making sure to throw the gel away in the gel waste bin.

Gel Purification

Reagents

Membrane Binding Solution	1 mL per each g of gel
Membrane Wash Solution	1200 uL
NF water	15 uL

Protocol

Weigh a microcentrifuge tube for each DNA fragment to be isolated and record weight
Use a UV lamp to excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade
Transfer the gel slice to the weighed microcentrifuge tube and record the weight
Subtract the weight of the empty tube from the total weight to obtain the weight of the gel slice
- The gel slice may be stored at 4°C or at –20°C for up to one week in a tightly closed tube under nuclease-free conditions before continuing purification
Add Membrane Binding Solution to the gel slice
Vortex the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved
Vortex the tube every few minutes to increase the rate of agarose gel melting
Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube
- Once the agarose gel is melted, the gel will not resolidify at room temperature
Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly
Incubate for 1 minute at room temperature before centrifugation for 1 minute and discarding supernatant
Wash with 700µL of Membrane Wash Solution, centrifuge for 1 minute, and discard supernatant
Wash with 500µL of Membrane Wash Solution, centrifuge for 5 minutes, and discard supernatant
Centrifuge 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol
Transfer SV Minicolumn to a new microcentrifuge tube
Add Nuclease Free water directly to the center of the column
- Membrane should be completely covered with nuclease-free water
Incubate at room temperature for 1 minute, centrifuge for 1 minute at 16,000 × g, and repeat
Discard the SV Minicolumn
Store the tube containing the eluted DNA at 4°C or –20°C
- Check the purity of DNA in the nanodrop
- If concentration not high enough, speed vac and nanodrop again

Gibson Cloning

Reagents:

DNA insert	3:1 insert to vector ratio. Should have 500 ng.
DNA vector	3:1 insert to vector ratio.
Gibson Master Mix	15 uL
NF H2O	Remaining volume necessary.
Total	20 uL

Procedure

Calculate the amount of insert, vector, and water needed. Use calculations on spreadsheets in drive.
- 3:1 insert to vector ratio
- Should have 500 ng of template DNA

Reagents	Concentration (ng/uL)	Concentration (g/uL)	# bp	Mol weight (amu)	M (Concentration/MW)	uL necessary for # fmol
Insert	given	Conc (ng/uL) * 10^(-9)	given	# bp * 650	Conc (g/uL) / mol weight	# fmol / M
Vector	given	Conc (ng/uL) * 10^(-9)	given	# bp * 650	Conc (g/uL) / mol weight	# fmol / M
Gibson Master Mix	n/a	n/a	n/a	n/a	n/a	15
NF H2O	n/a	n/a	n/a	n/a	n/a	20 - (sum)
				Total		20 uL

On ice, put the insert, vector, and water in a PCR tube with 15 uL of the gibson master mix.
- There should already be aliquots of 15 uL of gibson master mix (GA) in the fridge. Restock if not.
Run thermocycler program.

Golden Gate Cloning

Reagents

DNA inserts/parts	20 fmol DNA (per each insert/part) per 10 uL rxn. Calculations can be done with spreadsheet from Gibson calculations. Modify fmol calculations at end.
T4 Ligase Buffer 10X	1 uL buffer per 10 uL rxn
T7 Ligase	0.5 uL enzyme per 10 uL rxn
Type II Restriction Enzyme (BsaI or BsmbI)	0.5 uL enzyme per 10 uL rxn
NF H2O	Remaining volume necessary.

Protocol:

Calculate amount of reagents and DNA needed based on wanted volume of reaction and add all reagents to a PCR tube on ice, restriction enzyme last.
- 20 fmol DNA (for each insert/part) per 10 uL rxn
Run thermocycler program.

Transformation (electrotransformation w E. Coli)

Reagents:

Soc Medium	1 mL per 1 uL cells
DNA	~ 250 ng

Protocol:

Using electrocomp cells, let cells thaw on ice (a few minutes, do not leave out for longer than necessary). Keep cuvette and cuvette holder on ice as well.
Add DNA to cells once thawed. Need ~ 250 ng for transformation. Do not incubate.
Add cells to slit in cuvette, put cuvette in holder and push back into clasp of electroporator. Remember to press down on pulse until beep. Check time constant to make sure transformation successful.
Using sterile technique, add SOC medium to cuvette then immediately pipette draw SOC up with cells. Place in pre-prepared 1.5 mL tube.
Incubate cells in shaking incubator for 1 hr.
Afterwards, spin down at 3000 rpm for 5 min to pellet bacteria.
Take out SOC, but leave ~ 60 - 80 uLs. Resuspend pellet and plate.

Liquid Cultures

Reagents:

Nutrient Broth	4 mL
Antibiotic	1:1000 ratio between uL antibiotic and uL broth

Equipment:

Plate of Transformed Colonies
Large Tubes in Tube Rack
Bunsen Burner (sterile technique throughout)
Shaking Incubator

Protocol:

In front of the flame from the bunsen burner, put the nutrient broth and antibiotic in the large tube
- Superior broth has more nutrients than LB and therefore has a higher yield
- Antibiotic depends on antibiotic resistance of transformed colonies
Pick up a colony from the plate of colonies by touching the colony with a tip (on pipette)
When transferring the colonies to the large tubes, tilt the large tube so the nutrient broth is closer to the opening
Put the very end of the tip (which touched the plate and has the colony) into the broth
- Do not dip the entire tip in the nutrient broth because the tip is not that sterile
Shake the tip a little to transfer the colony into the nutrient broth
Take the very end of the tip out of the tube, discard the tube, and cap the tube
Put the tubes in a shaking incubator overnight
- Can either miniprep immediately or freeze culture tubes to miniprep later

Freezing Liquid Culture Tubes

Take liquid culture tubes out of the shaking incubator and centrifuge at 3000 rpm for 5 minutes
Dump the supernatant (liquid) into a beaker
Add 10% bleach to the beaker
Place the large tubes in the freezer covered with plastic wrap
- Take out the large tubes only when going to do the miniprep immediately

Miniprep

Purpose:
To purify plasmid from cells, usually with the liquid cultures created from a transformation

Reagents

Plasmid Resuspension Buffer (B1)	400 µL
Plasmid Lysis Buffer (B2)	400 µL
Plasmid Neutralisation Buffer (B3)	800 µL
Plasmid Wash Buffer 1	200 µL
Plasmid Wash Buffer 2	400 µL
DNA Elution Buffer	40 µL

Equipment:

Protocol:

(Based on Monarch Miniprep protocol with revisions from Michelle Byrom)

Digest

Reagents:

DNA	~ 250 ng in 2 uL
NF H2O	Remaining volume necessary to fill total volume.
Buffer	2 uL per enzyme
Enzyme	0.5 per enzyme
Total	20 uL

Protocol:

Add all reagents to a PCR tube on ice. Add enzyme last.
- Don’t forget to flick tube!!
Run thermocycler program.

(Incubation temperature) for 1 hr/overnight
(Inactivation temperature) for 20 min
4 C forever

Ligation

Reagents:

DNA	~ 250 ng in 2 uL
NF H2O	Remaining volume necessary to fill total volume.
T4 Ligase	2 uL per enzyme
Buffer	0.5 per enzyme
Total	20 uL

Protocol:

Add all reagents to a PCR tube.
Run thermocycler program.

60 C for 2 hr/overnight
4 C forever

LS/SB Broth

Reagents:

NF H2O	Dependent on objective.
Pre-mixed LB/SB Powder	Dependent on company.

Protocol:

Measure out water, add to bottle.
Using pre-mixed LB powder, add specified amount per liter. Mix with magnetic stir bar.
Take out stir bar.
Autoclave on liquid cycle for 20 min at 15 psi.

Glycerol Stocks

Reagents:

Liquid Culture	500 uL for 1 stock
50% Glycerol	500 uL for 1 stock

Protocol:

Inoculate liquid cultures using the standard protocol for liquid cultures and leave overnight or until there is bacterial growth.
Using sterile procedure. add 500 µL of the liquid culture and 500 µL of 50% glycerol to a 1.5 mL microcentrifuge tube. Close lid and gently mix by inverting the tube or vortexing to ensure that the culture and glycerol are not separated.
- Stock doesn’t always need to consist of 500 uL liquid culture and 500 uL glycerol, but liquid culture and glycerol must be in a 1:1 ratio.
Put the glycerol stock in the -80℃ freezer to freeze. Glycerol stock should be stable for years so long as it is left at -80℃. Subsequent thaw and freeze cycles will reduce shelf life.
To recover bacteria from a glycerol stock open the top and use a sterile hoop to scrape some frozen bacteria off the top. Streak onto a plate containing the appropriate antibiotic.
Grow on plates overnight at 37℃ (or a lower temperature like 30℃ if a slower growth rate is required).

Production of L-DOPA*

Reagents:

Liquid Cultures of Production Assembly and Controls	100 uL
NF H2O	Dependent on objective.
Pre-mixed Nutrient Powder (LB)	Dependent on objective.
Tyrosine	Enough to make 1 mM and 5 mM solution.

Protocol:

Make 3 different batches of LB Broth: plain LB, LB with 1 mM tyrosine, and LB with 5 mM tyrosine.
- Weigh out enough tyrosine to make 1 mM and 5 mM solution.
- Use standard LB protocol to make all three, with exception of adding tyrosine.
Use different batches of LB to make liquid culture stocks with production assembly as well as a control, another plasmid that doesn’t produce L-DOPA.
- Use already grown liquid cultures. Take 100 uL grown culture and add to 4 mL of LB/LB w 1mM tyrosine/LB w 5 mM tyrosine.
- Remember antibiotic.
- Make liquid cultures in triplicates.
Leave new culture tubes in shaking incubator.
Let cultures sit until turn dark (brown color).

Sensing and Reporting Dopamine*

Reagents:

LB Broth	Dependent on objective.
Dopamine	Enough to make 10 mM stock solution.
L-Ascorbic Acid	1 mg/uL solution.
Liquid Cultures of Sensing Assembly and Controls	100 uL
IPTG	Enough for 1 mM solution.

Protocol:

First make 10 mM DOPA stock solution in order to make other test conditions.
- Calculate mLs of 10 mM DOPA stock solution needed. Using sterile technique, add however many mLs of LB needed (for example, if 20 mLs of stock solution needed, add 20 mL LB and base calculations off of that) to a tube.
- Add enough dopamine to make 10 mM solution (continuing off example of 20 mL solution, add 0.0363 g of dopamine to make 10 mM solution).
- Add ascorbic acid in concentration of 1 mg/uL solution.
- Once all added, vortex tube for ~15 - 20 min until dopamine goes into solution.
- Sterilize solution by using a syringe and filter.
After having made 10 mM DOPA stock solution, make a 1:10 dilution of 10 mM stock solution to make a 1 mM solution. (If 20 mL of 10 mM stock solution, dilute 2 mL of 10 mM solution in 18 mL LB to make 20 mL of 1 mM DOPA solution). Remember to add ascorbic acid to new stock solution. Then sterilize like before.
Repeat this step until there is 10 mM, 1 mM, 100 uM, 10 uM, and 1 uM DOPA solution.
Use 1 mM, 100 uM, 10 uM, and 1 uM DOPA solution to make liquid cultures from already grown liquid cultures of sensing assembly and controls.
- Make 1:50 dilutions of already grown liquid cultures with different media conditions. For example, for 4 mL culture, add 80 uL of already grown culture to 4 mL of LB or a certain DOPA solution.
- Do triplicates for each condition for each assembly/control tested.
- Remember to make two identical sets, one without IPTG and one with.
Grow for ~ 6 hrs.
- When cell OD reaches .6, add 4 uL (1:10 of media) of 1 mM IPTG (after ~3 hours)
- Put cultures back into the shaking incubator
Spin down cultures
- ~3000 rpm for 5 minutes
- Pour out supernatant
Resuspend in 1x Buffer
- 4 mL of 1x PBS
- Pipette or vortex to resuspend cells
Plate reading
- Take 100 uL of the resuspended cells
- Load into a 96 well transparent plate
- Measure absorbance and fluorescence with Venus assay (excitation: 515 nm, emission: 528 nm)

Measuring Fluorescence*

Reagents:

Liquid Cultures	100 uL
PBS	5 mL

Protocol:

Liquid cultures
- Pick colonies from plate
- Multiple controls
- Overnight in the 37 incubator
Liquid cultures of already grown liquid cultures
- 100 uL of grown liquid cultures into 5 mL of media
- Remember antibiotic
- Incubate cultures for another 4-6 hours
Spin down cultures
- 3000 rpm for 5 minutes
Resuspend in 1x Buffer
- 4 mL of 1x PBS
- Pipette or vortex to resuspend cells
Plate reading
- Take 100 uL of the resuspended cells
- Load into a 96 well transparent plate
- Measure absorbance and fluorescence with GFP assay (excitation: 495 nm, emission: 509 nm)

Team:Austin UTexas LASA/Experiments

Experiments

Protocols

Reactants

Protocol