iGEM 2025 — Team hk-plknplc

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}This serves as a chronological record of your team's progress throughout
the season. It documents your daily activities, experiments, discussions, and
decisions.{% endblock %} {% block page\_content %}

## Months

* [June 2025](#june)
* [July 2025](#july)
* [August 2025](#august)
* [September 2025](#september)
* [October 2025](#october)

# Lab Notebook

Chronological lab notes — June → October 2025

## June 2025

25/06/2025

Preparation of kanamycin LB plates.

26/06/2025

Transformation of pET28a.

27/06/2025

Collection of plates.

![27/06/2025 plate collection](https://static.igem.wiki/teams/5884/all-photos/14.avif)

Figure — 27/06/2025: Plate collection results.

30/06/2025

Preparation of LB and SOB media.

## July 2025

02/07/2025

Prepared starter plate. Streak plate.

03/07/2025

Collection of starter plate.

![03/07/2025 starter plate](https://static.igem.wiki/teams/5884/all-photos/15.avif)

Figure — 03/07/2025: Starter plate collection.

04/07/2025

cultivate NiCo21 and DH5α overnight culture

05/07/2025

Harvest overnight culture of NiCo21 and DH5α

DH5α culture did not reach desired OD; NiCo21 overgrowth, all samples
are not able to proceed to the next step

Reasons: SOB contamination; increased ambient temperature (deviated
from usual 18°C); more vigorous shaking than expected.

08/07/2025

PCR of the newly arrived gene fragments from IDT

11/07/2025

Gel electrophoresis, smearing is found and will adjust the conditions
in the coming days

![11/07/2025 gel electrophoresis 1](https://static.igem.wiki/teams/5884/all-photos/16.avif)

Figure — 11/07/2025: Gel electrophoresis (smear observed).

![11/07/2025 gel electrophoresis 2](https://static.igem.wiki/teams/5884/all-photos/17.avif)

Figure — 11/07/2025: Gel electrophoresis (additional image).

17/07/2025

PCR. Preparation of medium.

18/07/2025

Competent cell preparation. Starter culture of DH5α showed a negative
result. NiCo21 gives positive results.

19/07/2025

Harvest overnight culture of NiCo21. Failed to proceed, the optical
density in 600nm wavelength of the bacteria culture exceed target OD
0.55.

21/07/2025

Preparation of materials: 800mL LB \*2 (total 1.6L); 500mL conical
flask \*6; 100mL tube \*2 box.

Gel electrophoresis of PCR products from 19/7.

DNA purification of same batch of PCR products.

23/07/2025

PCR to test conditions for the 6 gene fragments. Gel electrophoresis
of PCR products.

Preparation of overnight culture of NiCo21 and DH5α for competent cell
preparation. Both culture showed positive results before the lab end
on 23/7.

24/07/2025

Harvest overnight culture of NiCo21 and DH5α.

28/07/2025

Preparation of materials.

29/07/2025

Preparation of overnight culture of NiCo21 and DH5α.

30/07/2025

Harvest of cells. Perform DNA purification. Pick clones from the plate
made in 26/6 and inoculate an overnight culture for plasmid
preparation. Preparation of agar plates.

31/07/2025

Plasmid preparation. Gel electrophoresis to check results. Restriction
enzyme digestion.

## August 2025

02/08/2025

Stock check.

04/08/2025

DNA purification.

06/08/2025

DNA purification.

07/08/2025

Cultivation of overnight culture of NiCo21 and DH5α. PCR for the 6
fragments. DNA purification for the 6 fragment.

08/08/2025

Gel electrophoresis of the PCR products from 7/8. Proceed with the
preparation of competent cells, transparent solution, negative result
obtained.

11/08/2025

Cultivation of overnight culture.

12/08/2025

Proceed with the preparation of competent cells, prepared 20 tubes of
stock for each cells and stored in -80℃.

21/08/2025

Transformation of pET28a.

## September 2025

06/09/2025

Make Ligation product. Measure the concentration of the DNA in the
plasmid. prepare plasmid RE.

09/09/2025

Perform gel electrophoresis. Cut band.

10/09/2025

Gel purification. Transformation of the ligation product to E. Coli.

11/09/2025

Do gel electrophoresis. Check colonies in plates.

![11/09/2025 colonies](https://static.igem.wiki/teams/5884/all-photos/18.avif)

Figure — 11/09/2025: Colony check.

13/09/2025

Transformation pET28a to DH5α. Dilute pET28a. Prepare agarose gel.

![13/09/2025 transformation](https://static.igem.wiki/teams/5884/all-photos/19.avif)

Figure — 13/09/2025: Transformation setup.

Pet28a transformation. Perform RE and ligation. Check stock of diluted
pet28a. Dilute pet28a.

15/09/2025

Prepare Kan working solution. Check clones of transformed pet28a —>
no clones are presented (failed).

![15/09/2025 no clones](https://static.igem.wiki/teams/5884/all-photos/20.avif)

Figure — 15/09/2025: Plate check (no clones observed).

Check whether there are uncut MNEI, T1R2 and T1R3. Perform PCR.
Prepare LB. Transformation with newly diluted pet28a.

16/09/2025

Plasmid preparation. Check plasmid band. RE plasmid. PCR.

17/09/2025

Gel electrophoresis of 16/9 product and RE product.

![17/09/2025 gel electrophoresis](https://static.igem.wiki/teams/5884/all-photos/21.avif)

Figure — 17/09/2025: Gel electrophoresis image.

PCR Clean.

![PCR clean](https://static.igem.wiki/teams/5884/all-photos/22.avif)

Figure — PCR clean image.

Transformation. Plasmid preparation. PCR of IDT DNA. Prepare agarose
gel. Measure DNA concentration.

18/09/2025

Prepare agarose gels. Make Kan plates. Measure plasmid concentration.
Perform gel electrophoresis. Pick clone from the plates spreaded.

![18/09/2025 pick clones](https://static.igem.wiki/teams/5884/all-photos/23.avif)

Figure — 18/09/2025: Picking clones from plates.

Plasmid RE using PCR machine.

19/09/2025

Prepare agarose gels.

![19/09/2025 gels](https://static.igem.wiki/teams/5884/all-photos/24.avif)

Figure — 19/09/2025: Gel preparation and electrophoresis.

Perform gel electrophoresis on plasmid prep product on 18/9 (using 1
kbp ladder) and RE products (using 1k and 50bp) on 18/9. Prepare LB
medium. Take out the overnight culture for plasmid prep. Prepare
glycerol stock for pET28a. Plasmid purification. Elute the DNA from
the plasmid prep. Measure the DNA concentration of the eluted plasmid.

20/09/2025

Plasmid prep. Perform gel electrophoresis (plasmid prep products).
Perform gel purification (plasmid prep products). Check DNA
concentration of the plasmid prep. RE (1h elution incubation). Perform
electrophoresis (RE) & Gel purification (RE). Ligation (using gel
purification products).

22/09/2025

Prepare gel for gel purification (0.6%). Perform gel electrophoresis
for RE protocol. Switch the ligation condition from 4°C to 16°C (PCR
machine) and continue incubation. Transformation of 1:3 & 1:5 Ligation
product (MNEI Fragment 1-4, T1R2 & T1R3) to DH5a and NiCo 21 (Failed).
Perform gel purification. Elution. Quantification of the DNA
concentration. Ligation. Prepare 1L LB-agar. Make Kan plates. Perform
plasmid prep. Elution. Perform gel electrophoresis on plasmid prep
products. Check the concentration of the DNA in the plasmid prep
products.

25/09/2025

Prepare agarose gel.

![25/09/2025 gel](https://static.igem.wiki/teams/5884/all-photos/25.avif)

Figure — 25/09/2025: Gel for plasmid RE products.

Plasmid prep. Make Kan plates. Perform gel electrophoresis (RE
product).

![25/09 RE product](https://static.igem.wiki/teams/5884/all-photos/26.avif)

Figure — 25/09/2025: RE product gel image.

Gel purification (continue).

27/09/2025

Transformation of 1:3 & 1:5 Ligation product (MNEI Fragment 1-4, T1R2
& T1R3) to DH5a and NiCo 21 (Again). Plasmid prep (failed since no
bacteria appeared after centrifuge).

![27/09/2025 transformation attempt](https://static.igem.wiki/teams/5884/all-photos/27.avif)

Figure — 27/09/2025: Transformation attempt (no colonies).

Make RE. Make Kan plate.

29/09/2025

Pick clones of pET28a.

![29/09/2025 pick clones 1](https://static.igem.wiki/teams/5884/all-photos/28.avif)

Figure — 29/09/2025: Picked clones (image 1).

![29/09/2025 pick clones 2](https://static.igem.wiki/teams/5884/all-photos/29.avif)

Figure — 29/09/2025: Picked clones (image 2).

![29/09/2025 pick clones 3](https://static.igem.wiki/teams/5884/all-photos/30.avif)

Figure — 29/09/2025: Picked clones (image 3).

Perform gel purification. Measure DNA concentration of purified DNA
concentration.

![29/09/2025 DNA concentration](https://static.igem.wiki/teams/5884/all-photos/31.avif)

Figure — 29/09/2025: DNA concentration measurement.

30/09/2025

Pick clones of NiCo21.

![30/09/2025 NiCo21 photo 1](https://static.igem.wiki/teams/5884/all-photos/32.avif)

Figure — 30/09/2025: NiCo21 clone pick (1).

![30/09/2025 NiCo21 photo 2](https://static.igem.wiki/teams/5884/all-photos/33.avif)

Figure — 30/09/2025: NiCo21 clone pick (2).

Perform plasmid prep. Perform gel purification.

## October 2025

02/10/2025

Make 2 gels (one for MNEIs, one for T1R2/3). PCR of the prepped
plasmids and 2 RXN with just purified pET-28a. Plasmid prep for picked
clones. Transform if the band size is correct. Perform gel
electrophoresis.

![02/10/2025 gel image 1](https://static.igem.wiki/teams/5884/all-photos/34.avif)

Figure — 02/10/2025: Gel images (1).

![02/10/2025 gel image 2](https://static.igem.wiki/teams/5884/all-photos/35.avif)

Figure — 02/10/2025: Gel images (2).

![02/10/2025 colonies](https://static.igem.wiki/teams/5884/all-photos/36.avif)

Figure — 02/10/2025: Colony check for transformation done on 30/9.

03/10/2025

Perform plasmid purification. Make 2 gels (2%). Measure the DNA
concentration of transferred NiCo21 with 1:3 ligation products (MNEI
Fragment 1).

Transform N1 to NiCo21 (failed since someone used PCR products of N1
to transfer to NiCo21). PCR for purified plasmids.

04/10/2025

Prepare agarose gels. Perform gel electrophoresis for the PCR product.

![04/10/2025 image 1](https://static.igem.wiki/teams/5884/all-photos/37.avif)

Figure — 04/10/2025: Gel electrophoresis (1).

![04/10/2025 image 2](https://static.igem.wiki/teams/5884/all-photos/38.avif)

Figure — 04/10/2025: Gel electrophoresis (2).

![04/10/2025 image 3](https://static.igem.wiki/teams/5884/all-photos/39.avif)

Figure — 04/10/2025: Gel electrophoresis (3).

Transform the N1 to NiCo21. Perform RE + Ligation (1:3).

06/10/2025

Perform gel electrophoresis.

![06/10/2025 gel](https://static.igem.wiki/teams/5884/all-photos/40.avif)

Figure — 06/10/2025: Final gel images.

WIKI DONE

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

#### Silver Medal Criterion #1

Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.

---

Visit the [Medals page](https://competition.igem.org/judging/medals) for more information.

## Tips for Success

---

Engineering success can be achieved by documenting your effort to follow the engineering design cycle: Design → Build → Test → Learn

We invite you to think about ways to tackle and solve one or more of your project's problems and use synthetic biology tools and/or experimental techniques to generate expected results.

When you have completed the cycle once, think about and document what changes in design you would make for the next iteration(s) of the cycle.

For example, you can design and build a new Part, measure its performance, document whether it worked or not, and propose how the results would inform the next design or steps (documentation must be on the Part's Pages on the Registry).

Visit the [Engineering pages](https://technology.igem.org/engineering) for additional guidance on engineering success.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}**Step-by-Step Experimental Design** Our journey through lab work and discovery{% endblock %}
{% block page\_content %}
{% include 'menu.html' %}

## Experiments

* [Preparation of Reagents](#preparation)
* [PCR Amplification](#pcr)
* [DNA Purification](#dna)
* [Plasmid Purification](#plasmid)
* [Agarose Gel Electrophoresis](#gel)
* [Preparation of Competent Cells](#competent)
* [Transformation](#transformation)
* [Restriction Digestion](#digestion)
* [Ligation](#ligation)

## Preparation of Reagents

**Table 1. materials of SOB medium per liter:**

| Component | Amount |
| --- | --- |
| Yeast Extract | 5 g |
| Tryptone | 20 g |
| KCl | 0.186 g |
| MgCl₂ | 3.808 g |
| NaCl | 0.584 g |

**Table 2. materials of LB medium (liquid/agar) per liter:**

| Component | Amount |
| --- | --- |
| Yeast Extract | 5 g |
| Tryptone | 10 g |
| NaCl | 10 g |
| Agar (for solid medium) | 20 g |

**Preparation of LB/SOB medium Steps:**

1. Take an empty reagent bottle and rinse it with milliQ water
2. Use electronic balance, weighting boat and a spatula to add the required amounts of materials into the reagent bottle
3. Add water to the volume needed
4. Close the lid of the reagent bottle and autoclave it at 121°C for 15-20 minutes

## PCR Amplification

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

| Component | 25µL Reaction | 50µL Reaction (for gel extraction) |
| --- | --- | --- |
| Q5 High-Fidelity 2× Master Mix | 12.5 µL | 25 µL |
| Forward Primer (10 µM) | 1.25 µL | 2.5 µL |
| Reverse Primer (10 µM) | 1.25 µL | 2.5 µL |
| Template DNA | 20 ng | 20 ng |
| Nuclease-Free Water | to 25 µL | to 50 µL |

2. Set the PCR Thermocycling Conditions:

| Step | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 98°C | 30 sec |
| 35 Cycles | 98°C | 10 sec |
| 35 Cycles | 68  70.5°C | 30 seconds (for MNEIs and TAS1R3)  30 seconds (for TAS1R2) |
| 35 Cycles | 72°C | 10 seconds (for MNEIs)  25 seconds (for TAS1R2/3) |
| Final Extension | 72°C | 2 min |
| Hold | 4°C | ∞ |

## DNA purification

**DNA Fragment purification**

1. Add a 3X volume of Buffer DC to the PCR or other enzymatic reactions.
2. If the required volume of Buffer DC is less than 100 μL, add 100 μL of Buffer DC.
   Vortex briefly to mix the sample.Place a Spin Column into a Collection Tube.
3. Pipette the sample from Step 1 into the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through.
4. Note: For improvement the recovery of DNA, transfer the flow-through to Spin Column and centrifuge again.Add 700 μL Buffer WB to Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Dis the flow-through.
5. Note: Make sure that the volume of 100% ethanol indicated on the bottle label has been added to the Buffer WB.Repeat Step 4.
6. Place the Spin Column back into the Collection Tube. Centrifuge at 12,000 rpm for 1 minute.
7. Place the Spin Column into a new clean 1.5 ml tube. Add 25 −30 μL Elution Buffer or sterilized water to the centre of the membrane.
8. Let it stand for 1 minute at room temperature.
9. Note

* Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
* For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.

10. Centrifuge at 12,000 rpm for 1 minute at room temperature to elute DNA.

## Plasmid purification

1. Growth of bacterial cultures. Pick a single colony from a freshly streaked selective plate into 1 - 4 mL of liquid culture containing the appropriate selective antibiotic. Incubate at 37℃ overnight with vigorous shaking. (Growth for 12 - 16 hours and do not exceed 16 hours, otherwise the bacterial cells will be hard to lyse and the yield of plasmid DNA will be reduced.)
2. Note: The culture volume should not be excessive because excessive bacteria will decrease lysis efficiency and result in poor purity of plasmid DNA.Use 1 - 4 mL of the E. coli culture. Centrifuge at 12,000 rpm for 2 minutes to harvest the cell. Dis the supernatant.
3. Add 250 μL Solution l (containing RNase A). Resuspend the bacterial cell pellet completely by pipetting up and down.
4. Note: Be sure that the bacteria are completely resuspended, and no cell clumps remain before addition of Solution ll.Add 250 μL Solution ll and mix gently by inverting the tube 5 - 6 times to completely lysis the cell until the solution becomes viscous and slightly clear.
5. Note: Do not allow the lysis reaction to proceed more than 5 minutes.Add 350 μL of 4℃ precooling Solution lll and mix immediately and thoroughly by inverting the tube 5 - 6 times until a compact white pellet has been formed. Incubate at room temperature for 2 minutes.
6. Centrifuge at 13,200 rpm at room temperature for 10 minutes.
7. Note: Centrifuging at 4℃ is not recommended for precipitation.Apply the supernatant from Step 6 into a new clean 1.5 mL tube by precise pipetting. Centrifuge at 13,200 rpm at room temperature for 10 minutes.
8. Note: The white precipitate will clog the spin column, reducing the yield.Place a Spin Column in a Collection Tube.
9. Apply the supernatant from Step 6 onto the Spin Column by pipetting. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
10. Pipette 500 μL of Buffer WA onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
11. Pipette 700 μL of Buffer WB onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Dis the flow-through with the aid of pipette.
12. Note: Make sure that the amount of 100% ethanol indicated on the bottle label has been added to Buffer WB.Repeat Step 11.
13. Place the Spin Column back into new 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Dis the flow-through with the aid of pipette.
14. Place the Spin Column back into 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Dis the flow-through with the aid of pipette.
15. Note: Residual ethanol from Buffer WB may inhibit subsequent enzymatic reaction.Place the Spin Column in a new clean 1.5 mL tube. Add 50 μL Elution Buffer or sterile purified water to the center of the Spin Column membrane. Incubate for 1 and a half hour at 37℃. Note: Pre-heat Ultra-purified wter to 60℃
16. Centrifuge at 13,200 rpm for 1 minute to elute DNA.

## Agarose Gel Electrophoresis

1. Measure 0.2g of agarose.
2. Mix agarose powder with 20 mL 1xTAE in a microwavable flask.
3. Microwave until the agarose is completely dissolved.
4. Dilute the GelGreen® 10,000X stock reagent into the molten agarose gel solution at 1:10,000 and mix thoroughly. GelGreen® can be added while the solution is still hot.
5. Cast the gel and allow it to solidify. Load samples and run the gels (90V, 30 mins).
6. Image the stained gel with a 254 nm UV transilluminator or 490nm to 500nm visible light imaging system with a long path green filter.

## Preparation of NiCo21/DH5-ɑ Competent Cell

Prepare Inoue transformation buffer (chilled to 0°C before use).

1. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by
   dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent).
2. Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable pre-rinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C.

## Preparation of Inoue transformation buffer

1. Dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O.

**2. Reagent Amount per liter Final concentration:**

| Reagent | Amount per liter | Final concentration |
| --- | --- | --- |
| MnCl2•4H2O | 10.88g | 55 mM |
| CaCl2•2H2O | 2.20g | 15mM |
| KCl | 18.65g | 250 mM |
| PIPES (0.5 M, pH 6.7) | 20mL | 10 mM |

2. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C.
3. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 mL of SOB medium (LB may be used instead) in a 250-mL flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm).
4. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 mL of SOB. The first flask receives 10 mL of starter culture, the second receives 4 mL, and the third receives 2 mL. Incubate all three flasks overnight at 18-22°C with moderate shaking.
5. The following morning, read the OD600 of all three cultures.Continue to monitor the OD every 45 minutes.
6. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Dis the two other cultures.
7. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for10 minutes at 4°C.
8. Pour off the medium and store the open centrifuge bottle on a stack of paper towels
   for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
9. Resuspend the cells gently in 80 mL of ice-cold Inoue transformation buffer.
10. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C.
11. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.
12. Resuspend the cells gently in 20 mL of ice-cold Inoue transformation buffer.
13. Add 1.5 mL of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
14. Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed.
15. When needed, remove a tube of competent cells from the -70°Cfreezer. Thaw the
    cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the
    tube to an ice bath. Store the cells on ice for 10 minutes.
16. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Include all of the appropriate positive and negative controls.
17. Add the transforming DNA (up to 25 ng per 50 µl of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times
    to mix their contents. Set up at least two control tubes for each transformation
    experiment, including a tube of competent bacteria that receives a known amount of
    a standard preparation of superhelical plasmid DNA and a tube of cells that receives
    no plasmid DNA at all. Store the tubes on ice for 30 minutes.
18. Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
19. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 minutes.
20. Add 800 µl of SOC medium to each tube. Warm the cultures to 37°C in a waterbath, and then transfer the tubes to a shaking incubator set at 37°C. Incubate the cultures for 45 minutes to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
21. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic.
22. Store the plates at room temperature until the liquid has been absorbed.
23. Invert the plates and incubate them at 37°C. Transformed colonies should appear in 12-16 hours.

## Transformation

**Bacteria Transformation**

1. Thaw competent *E. coli* cells on ice.
2. Add 1–4 ng plasmid DNA and incubate 30 minutes.
3. Incubate for 30 minutes on ice.
4. Heat shock at 42℃ for 45 seconds.
5. Incubate for 30 minutes on ice.
6. Add LB medium so total volume is 1 mL.
7. Recover cells for 90 minutes in a shaker at 37°C at 270 r.m.p.
8. Centrifuge cells at 3000xg for 3 minutes.
9. Remove all liquid medium from the tube.
10. Resuspend in 100 ul of LB medium.
11. Spread plates using glass plates.
12. Add 6 beads on either side of each plate.
13. Flicker tube until cells are uniformly distributed.
14. Gently pipette a dilution of outgrowth onto the centre of the pre-warmed plate.
15. Vigorously move the plates forward and backwards. Make two complete 360° rotations, with stops every 90 degrees.
16. Allow plates to sit for 2 minutes.
17. Pour off the beads into a beaker.
18. Incubate plates at 37°C.

## Restriction Enzyme Digestion

1. Transfer contents into a 0.2mL PCR tube according to the following recipe:

| Component | 50µL Reaction |
| --- | --- |
| DNA | 1 µL |
| 10X CutSmart Buffer | 5µL (1X) |
| NcoI-HF | 1.0µL (20 units) |
| XhoI | 1.0µL (20 units) |
| Nuclease-free Water | to 50 µL |

2. Heat inactivation (65°C for EcoRI-HF® & 80°C for NheI-HF®)

## Ligation

1. Transfer contents into a 1.5 mL microcentrifuge tube according to the following recipe on ice:

| Component | 20µL reaction |
| --- | --- |
| T4 DNA Ligase Buffer (10×) | 2 µL |
| Vector DNA | varies |
| Insert DNA | varies |
| T4 DNA Ligase | 1 µL |
| Water | Top up to 20 µL |

2. Use NEBioCalculator to calculate molar ratios.
3. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
4. Gently mix the reaction by pipetting up and down and microfuge briefly.
5. Incubate at 16°C overnight

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Clearly and objectively describe the results of your experiments and research.
* Use tables, graphs, and images to visualize your data.
* Discuss the implications of your findings in the context of your project goals.
* Highlight any trends, patterns, or significant observations.
* Include an analysis summary section to synthesize your findings and discuss their overall significance.
* Outline your future plans for the project, including potential applications, further research, and development.
* Explain what went wrong and what you learned from these experiences.
* Discuss potential improvements or alternative approaches.
* Emphasize the importance of scientific honesty and transparency.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

{% endblock %}