Team:BFSUICC-China/Notebook
Construct the circuit
MAKING LB AGAR PLATES
1.Add 250 mL of dH2O to a graduated cyclindar.
2.Weigh out,： 5.0 g tryptone；2.5 g yeast extract；5.0 g NaCl；7.5 g agar
3.Mix powder well to bring into solution
4.Add dH2O to total volume of 500 mL and transfer to 1 L flask
5.Put on stirring hot plate and heat to boil for 1 min while stirring.
6.Transfer to 1 L pyrex jar and label with autoclave tape.
7.Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
8.Let agar cool
Pouring the Plates
1.Make sure bench has been sterilize
2.Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover off excessively
3.Swirl plate in a circular motion to distribute agar on bottom completely.
4.Store plates in plastic bags in fridge with: name, date and contents (note any additive).
BACTERIAL TRANSFORMATION
1.Take competent cells out of -80°C and thaw on ice
2.Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3.Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
4.Mix 10 μl of DNA into 50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
5.Incubate the competent cell/DNA mixture on ice for 30 mins.
6.Heat shock each transformation tube by placing the bottom 2/3 of the tube into a 42°C water bath for 60 secs.
7.Put the tubes back on ice for 2 min.
8.Add 450 μl LB (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
9.Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
10.Incubate plates at 37°C overnight.
INCUBATE BACTERIAL CULTURE OVERNIGHT
1.Prepare liquid LB.
For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract ddH2O to 400 mL
2.When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration (see table below).
3.Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
4.Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
5.Loosely cover the culture with sterile aluminum foil.
6.Incubate bacterial culture at 37°C for 12h in a shaking incubator.
7.After incubation, check for growth, which is characterized by a cloudy haze in the media.
EXTRACT PLASMID DNA
1.Pellet 4 ml bacterial overnight culture by centrifugation at 13000 rpm for 3 min at room temperature.
2.Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube.
3.Add 250μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear.
4.Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 6 times.
5.Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
6.Apply the supernatant from step 5 to the QIAprep spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through
7.Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
8.Centrifuge for 60 s and discard the flow-through.
9.Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for60 s and discard the flow-through.
10.Centrifuge for 1 min to remove residual wash buffer.
11.Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50μl water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
OPTIMIZING RESTRICTION ENDONUCLEASE REACTIONS
PstⅠ-HF
Reaction Condition CutSmart Buffer, 37℃
SpeⅠ
Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 80℃ 20min
XbaⅠ
Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 65℃ 20min
EcoRⅠ
Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 65℃ 20min
AGAROSE GEL ELECTROPHORESIS
Pouring a Standard 1% Agarose Gel:
1.Measure 1 g of agarose.
2.Mix agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe.
3.Microwave for 1-3 min until the agarose is completely dissolved.
4.Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
5.Pour the agarose into a gel tray with the well comb in place.
6.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.
Loading Samples and Running an Agarose Gel:
1.Add loading buffer to each of your DNA samples.
2.Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3.Fill gel box with 1xTAE until the gel is covered.
4.Carefully load a molecular weight ladder into the first lane of the gel.
5.Carefully load your samples into the additional wells of the gel.
6.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.
7.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8.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.
EXTRACTION THE DNA FRAGMENT FROM THE AGAROSE GEL
1.Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2.Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl).
3.Incubate at 50°C for 10 min (or until the gel slice has completely dissolved).To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
4.After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5.Add 1 gel volume of isopropanol to the sample and mix.
6.Place a QIAquick spin column in a provided 2 ml collection tube.
7.To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
8.Discard flow-through and place QIAquick column back in the same collection tube.
9.Recommended: Add 0.5ml of Buffer QG to QIAquick column and centrifuge for1min.
10.To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
11.Discard the flow-through and centrifuge the QIAquick column for an additional 1min at 13,000 rpm.
12.Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13.To elute DNA, add 50µl water (pH7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1min. Alternatively, for increased DNA concentration, add 30µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
14.If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
T4 LIGASE
1.Set up the following reaction in a microcentrifuge tube on ice.
2.Gently mix the reaction by pipetting up and down and microfuge briefly.
3.Incubate at 16°C overnight or room temperature for 10 minutes.
4.Heat inactivate at 65°C for 10 minutes.
5.Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
AGAROSE GEL ELECTROPHORESIS（checking）
we run the electrophoresis again to check the base pair is correct or not, thus we could know our ligation is successful or not.
Test the function of parts and circuit
Plate Reading (Relative and Absolute Fluorescence)Day 1 :
Day 2 :
Prepare Copper ion solution in different concentration
Prepare Copper ion solution in different concentration
Transfer copper ion solution and bacteria of Abs 600 of 0.1 in LB medium into 2ml deep-well plates:
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