Team:OhioState/Experiments

Biobrick DNA Extraction

To use the DNA in the Distribution Kit, follow these instructions: ,/p>

Note: There is an estimated 2-3ng of DNA in each well. When following this protocol, assume that you are transforming with 200-300pg/µL

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
  2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
  3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
  4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
  5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Qiagen Genomic DNA Purification - DNA QIAamp DNA Mini Blood Mini Handbook

Cut up to 25 mg tissue into small pieces, place in a 1.5 mL microcentrifuge tube, and add 180 uL of Buffer ATL. Add 20 uL of proteinase K, vortex, then incubate at 55 C until the tissue is completely lysed (in water bath, at least 2 hours, can be overnight).

Vortex for 15 seconds, add 200 uL of Buffer AL to the sample, vortex again, incubate at 70 C for 10 minutes. Add 200 uL of EtOH (96-100%) to the sample, then vortex.

Pipet the mixture from step 4 into DNeasy Mini spin column placed in a 2-mL collection tube. Centrifuge at 8000 rpm for 1 min. Discard flow-through and collection tube.

Place spin column in a new 2-mL collection tube, add 500 uL of Buffer AW1, centrifuge for 1 min @ 8000 rpm. Discard flow-through and collection tube.

Place spin column in 2-mL collection tube, add 500 uL of Buffer AW2, centrifuge for 3 min @ 14000 rpm to dry the membrane. Discard flow-through and collection tube.

Place spin column in clean microcentrifuge tube, pipet 200 uL of Buffer AE directly onto membrane. Incubate at room temp for 1 min, then centrifuge for 1 min @ 8000 rpm.

Qiagen QIAprep Spin Miniprep Kit

  1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
  8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube.
  9. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Mach1 OneShot Chemical Transformation Protocol

  1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle (). For vacuum manifold processing, follow the instructions marked with a circle (). Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
  8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube.
  9. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Addgene Agarose Gel Electrophoresis Protocol

Measure 1 g of agarose.

Double to obtain 2% agarose gel

Add agarose to 100 mL 1x TAE buffer in a microwavable flask.

TBE can substitute for TAE.

Microwave 1-3 minutes until completely dissolved.

Take it out and swirl every 30-45 seconds. Beware surprise boiling. Stop microwaving as soon as boiling occurs; excessive evaporation will change the agarose percentage. Don't let it boil over.

Cool to about 50 °C (about when it is comfortable to keep your hand on the flask), about 5 minutes.

Add ethidium bromide (EtBr) to a final concentration of xxx (usually about 10 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.

Pour slowly into a gel tray with the well comb in place.

Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.

Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified

Add loading buffer to each of your DNA samples.

Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.

Once solidified, place the agarose gel into the gel box (electrophoresis unit). Fill gel box with 1xTAE (or TBE) until the gel is covered.

Carefully load a molecular weight ladder into the first lane of the gel. Carefully load your samples into the additional wells of the gel.

Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.

Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

IDT Primer Resuspension Protocol

Briefly centrifuge the tubes before opening them.

Resuspend in dH2O to 100 uM.

Vortex briefly to ensure complete dissolution, then spin with a small tabletop microcentrifuge for a few seconds to pull the liquid back to the bottom of the containers.

Dilute an aliquot with dH2O to 10 uM.

NEB 2-Step PCR

Initial Denaturation

98 C for 30s

30-35 Cycles

98 C for 5-10s

72 C for 30-50s/kb

Final Extension

72 C for 5-10min

Biobrick Assembly - Digestion

To assemble an Upstream Part with a Downstream Part into a Destination Plasmid

Digest Upstream Part with EcoRI-HF® and SpeI.

Upstream Part Plasmid:

500 ng

EcoRI-HF:

1 µl

Spel:

1 µl

10X NEBuffer 2.1:

5 µl

H2O:

to 50 µl

Digest Downstream Part with XbaI and PstI.

Upstream Part Plasmid:

500 ng

Xbal:

1 µl

Pstl:

1 µl

10X NEBuffer 2.1:

5 µl

H2O:

to 50 µl

Digest the Destination Plasmid with EcoRI-HF® and PstI: The Destination Plasmid DNA should either be prepared with PCR or contain a toxic gene (e.g. ccdB, sacB) in the cloning site to avoid the need for gel purification. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

Destination Plasmid DNA:

500 ng

EcoRI-HF:

1 µl

Pstl:

1 µl

10X NEBuffer 2.1:

5 µl

H2O:

to 50 µl

Incubate all three restriction digest reactions at 37°C for 10 (15) minutes and then heat inactivate at 80°C for 20 minutes.

Biobrick Assembly - Ligation

Ligate the Upstream and Downstream Parts into the digested Destination Plasmid

Upstream Part digestion:

2 µl

Downstream Part digestion:

2 µl

Destination Plasmid digestion:

2 µl

10X T4 DNA Ligase Buffer:

2 µl

T4 DNA Ligase:

1 µl

H2O:

11 µl

Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Transform 2 μl of the ligation product into 50 μl of competent E. coli cells (or other suitable host strain). Select using the antibiotic corresponding to the Destination Plasmid.

Gibson Assembly

Materials: 

  • gblock (resuspended).
  • Linearized vector backbone.
  • NEBuilder HiFi DNA Assembly Master Mix. 
  • H2O. 
  • Positive control. 

Procedure:

  1. Use the fragment assembly protocol from the HiFi DNA Assembly table. 
  2. Each insert must be at least twice the vector. For this reaction – Vector = 0.05 pmol and each Insert = 0.1 pmol. Allowing the total reaction to be under 0.5 pmol. 

    Reagent Volume (µl) pSB1C3 backbone: 2.35 BBa_K2797002: 5.74 Master Mix: 8.09 

  3. Add the fragments and vector to a PCR tube with the master mix and water if required. 4. Incubate samples on a heat block 60 minutes at 50 °C.

Electroporation Protocol + Mannitol Glycerol Electrocompetent Wash 

*On the 5430R Centrifuge Maxspeed 

  1. Take 5 mL of culture and extract 1 mL
  2. Spin this down and resuspend with 350 µl chilled water.
  3. Insert 250 µl M-G at the bottom to form the bottom layer
  4. Spin this down, remove the top and then bottom layer
  5. Resuspend in 100 µl M-G solution
  6. Add 100-1000 ng DNA
  7. Shock and grow in SOC media, plate on LB+cam plates

Nitrogen Free Media

Ingredients

Gms/litre

Sucrose

20.000

Dipotassium phosphate

1.000

Magnesium sulphate

0.500

Sodium chloride

0.500

Ferrous sulphate

0.100

Sodium molybdate

0.005

Calcium carbonate

2.000

Nitrogen-Deficient Media

To Nitrogen-Free media add 2mM Glutamate

Nitrogen Enriched Media

To Nitrogen-Free media add 100 mM NH4Cl

Autoclave liquid cycle for 1 hour. Some particulate will not go into solution.

@igem.osu

@igem_osu

igem.osu@gmail.com

IGEM at Ohio State