iGEM 2018 — Team Tacoma_RAINmakers

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tacoma RAINmakers/Notebook

Team:TacomaRAINmakers/Notebook - 2017.igem.org

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:ECUST/Lab/Notebook

[![](https://static.igem.org/mediawiki/2018/3/3a/T--Tacoma_RAINmakers--teamlogo.png)](https://2018.igem.org/Team%3ATacoma_RAINmakers)

* [HOMEPAGE](https://2018.igem.org/Team%3ATacoma_RAINmakers)
* [Team](https://2018.igem.org/Team%3ATacoma_RAINmakers/Team)
  + [Sponsors](https://2018.igem.org/Team%3ATacoma_RAINmakers/Sponsors)
* [Project](https://2018.igem.org/Team%3ATacoma_RAINmakers/Description)
  + [Experiments](https://2018.igem.org/Team%3ATacoma_RAINmakers/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATacoma_RAINmakers/Notebook)
  + [Interlab](https://2018.igem.org/Team%3ATacoma_RAINmakers/InterLab)

  + [Attributions](https://2018.igem.org/Team%3ATacoma_RAINmakers/Attributions)
* [Parts](https://2018.igem.org/Team%3ATacoma_RAINmakers/Parts)
* [Safety](https://2018.igem.org/Team%3ATacoma_RAINmakers/Safety)
* [Human Practices](https://2018.igem.org/Team%3ATacoma_RAINmakers/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3ATacoma_RAINmakers/Public_Engagement)
* [Awards](https://2018.igem.org/Team%3ATacoma_RAINmakers/Awards)
* [Judging Form](https://igem.org/2018_Judging_Form?team=Tacoma_RAINmakers)

Team- 2018.igem.org

# Team:RAINmakers/Notebook

# Tacoma RAINmakers Lab Notebook

# Week 1

## Digestion and Isolation of pSB1C3 Backbone.

The purpose of digesting the pSB1C3/PArsRGFP construct was to separate the backbone from the former GFP insert. Tacoma RAINmakers sought to isolate the pSB1C3 backbone employed in their 2017 construct, as the GFP reporter complex was no longer desired. Apparent disadvantages of GFP indication in biosensors are ultraviolet readings. The RAINmakers prefered a chromoprotein that produces color in the visible spectrum. Enzymes XbaI and SpeI were used to cleave the terminator sites of the vector, freeing the pSB1C3 backbone. NEB resources confirmed that the Cutsmart Buffer 2.1 is the most compatible with these particular enzymes, providing an optimized environment for digestion. Combining the reagents listed in Table 1.0, the reaction was set at 37ºC in a water bath for 1 hour and 45 minutes. This reaction was completed in duplicate to increase statistical probability of desired backbone DNA.

Following completion of pSB1C3 digestion, Tacoma RAINmakers employed a standard procedure listed in the protocol page as “Agarose Gel Electrophoresis.” The purpose of this gel was to confirm if the backbone DNA had been successfully isolated from the undesired GFP insert. As cited in Figure 1, the expected pSB1C3 bands were located at 2000bp. A gel extraction process, also outlined in the protocol section, was completed in order to remove and contain the digested pSB1C3 DNA.

![](https://static.igem.org/mediawiki/2018/e/e0/T--Tacoma_RAINmakers--Week1Figure1.png)
*Fig. 1. DNA gel of digested parts.*

# Week 2

## Digestion and Ligation of spisPink, amilCP, and PcArsR Inserts

Using IDT stock solutions of chromoprotein and arsenic regulatory DNA, Tacoma RAINmakers completed a standard restriction digest (see protocol page for further information). The notable difference between insert and backbone digestion are the enzymes. Each insert included a SalI enzyme site, which is not compatible with the BioBrick suffix/prefix of the vector. Instead, the SalI site is used to ligate the chromoprotein to PcArsR, rendering the ends of this complete insert as compatible for sticky-end ligation to pSB1C3. After combining reagents listed in Table 2.0, reaction was held in a water bath at 37°C overnight.

Once the SalI site in spisPink, amilCP, and PcArsR was successfully sticky-ended, Tacoma RAINmakers were prepared for ligation of each chromoprotein to the arsenic regulator. 60ng of each part were used in order to ensure that there would enough DNA material for the ligation. Having combined the substances from Table 2.1, the reaction was set to ligate overnight at 16°C. Afterwards, SalI was heat inactivated at 80°C, ensuring a complete denature, since the enzyme was no longer required.

# Week 3

## Digestion and Ligation of Insert (amilCP/spisPink + PcArsR) to pSB1C3 Backbone

Following the ligation of the chromoprotein and PcArsR, the complete insert was digested with enzymes compatible with our pSB1C3 backbone. This process allows sticky-ended ligation in the next step, which increases the chance of a proper insert-backbone ligation. 84 ng of insert DNA was pipetted into the reaction alongside the other reagents mentioned in Table 3.0. The digestion was set at 37°C in a water bath for 1 hour and 30 minutes.

Tacoma RAINmakers combined the reagents listed in Table 3.1 to ligate the completed insert to the vector. The reaction included a negative control that contained only vector DNA. A notable process involved in ligation reactions is calculating DNA volumes. Typically, a 1:3 ratio of vector to insert ensures that there is a balance of both parts. The RAINmakers employed the NEBioCalculator to determine how many moles were in 1ng of vector (2070bp) and 1ng of insert (1488bp). This calculation translated to 0.8µL of vector and 20µL of insert. The ligation occurred at 16ºC overnight and was heat inactivated at 80ºC for 20 minutes the following morning.

## Initiation of Individual Chromoprotein and Regulator Plasmid Design

A standard procedure in the Tacoma RAINmakers project is PCR amplification. This process is listed under the protocol page as “Insert PCR Amplification.” In preparation for ligation of inserts (amilCP, spisPink, and PcArsR) into the vector, all insert DNA must be amplified from its original limited stock. Once the PCR reaction has exited the thermocycler, gel electrophoresis must be employed to assess the efficacy of the amplification. As pictured in Figure 3, both the spisPink and amilCP bands successfully appeared at about 1000bp, and the PcArsR expressed at about 550bp. Unfortunately, the PcArsR negative control produced DNA bands, which suggested contamination during the PCR amplification process.

Following a successful PCR amplification confirmed by the gel, Tacoma RAINmakers performed a standard gel extraction. With much more insert DNA, RAINmakers were prepared to design three additional plasmids containing single inserts for isolated testing.

![](https://static.igem.org/mediawiki/2018/8/82/T--Tacoma_RAINmakers--Week3Figure1.png)
*Fig. 2. DNA gel of PCR products.*

# Week 4

## Transformation of Arsenic Construct and PCR Screening

TThe positive control (pSB1C3 + Insert) and negative control (pSB1C3 only) were transformed using DH5 Alpha Competent E. coli cells. Although the iGEM protocol for transformation states that 1µL of DNA is sufficient, 2µL of DNA were used for both the positive and negative control plates to ensure enough DNA existed for multiple colonies to grow. Transformation reactions were incubated overnight at 37ºC within a 14-18 hour time period. An extended description of the standard transformation process is listed under the protocol page.

The RAINmakers ran a PCR screening for both the positive (vector and insert) and negative (vector only) controls. This PCR allows us to amplify our cloned DNA, which is necessary to do because it will help us determine whether or not the ligation of the vector and insert was successful. The product after our PCR will be used to run a gel, which will help us see which colonies have the vector and insert and which only have the vector. After determining the volumes of the reagents, we decided to add an extra 20% uncertainty to each reagent in the master mix since small volumes of liquid can get stuck in the pipette. We did all these calculations based on the fact that we planned on doing 8 PCR reactions (8 PCR tubes). Before beginning the process of adding all the reagents to our PCR tubes, we needed to determine how long the extension part of PCR should be based on the base pair count of our product. Running a PCR simulation of Snapgene allowed us to find the exact base pair count of our positive control, which is 1597 base pairs. From this, we found that the extension period should be 1 minute and 36 seconds.

After adding all of our reagents together into 8 PCR tubes, we needed to select single colonies from transformation plates from 6/19/18. We determined that 6 of our PCR tubes would have the positive control, 1 would be the negative control and the last tube would have no DNA. Once we selected a single colony and put the DNA into a PCR tube, we streaked it onto a new plate (cut up in 8 different sections for each 8 different colonies) and labeled them 1, 2, 3, 4, 5, 6 (positive control), 7 (nothing) and 8 (negative control). We then incubated this new plate so that more colonies would grow. Finally, we put our PCR tubes in a thermocycler and ran our PCR.

## Blunt-End Digestion and Ligation of pSB1C3 and Inserts

Designing plasmids with individual inserts becomes slightly complicated, as each insert contains a SalI site that is not compatible with the BioBrick prefix/suffix of the backbone. Hence, Tacoma RAINmakers preferred blunt-end ligation, rendering the incompatible ends irrelevant.

Additionally, each insert was digested with its respective enzyme and filled in with T4 polymerase in preparation for blunt-end ligation.

# Week 5 - Week 7

We re-started the cloning process with fresh PCR products and more vector. We re-ligated our pSB1C3 backbone with our PcArsR+SpisPink/PcArsR+amilCP insert several times with only negative results. We decided to re-ligate PcArsR + chromoprotein (spisPink or amilCP), as there were speculations that something went wrong in the initial ligation of the two. This ligation contained 3 uL of PcArsR, 3 uL of chromoprotein (spisPink or amilCP), 1x T4 DNA Ligase Buffer, 1.5U T4 DNA Ligase, and molecular water to a final volume of 10 uL. We ligated at 16ºC overnight, then heat killed at 80ºC for 20 minutes.

After this ligation was complete, we digested the newly ligated insert with XbaI and SpeI to get it ready for ligation with the pSB1C3 backbone. This digestion contained 1 uL of Buffer 2.1, 1 uL of XbaI, 1 uL of SpeI, and the newly ligated insert DNA. We did an overnight transformation and then ran PCR on the colonies. To see the results we ran it on a 1% agarose gel and saw that the insert did not ligate to the vector.
This sequence of events repeated itself for many weeks, to great frustration.

# Week 8

## Restart cloning process

After failed attempts to clone the circuit, we decided to start again from the beginning. In the digestion of the backbone, we followed the iGEM protocol, but modified it by doing an overnight digestion at 16ºC instead of a 30 minute digestion. The same digestion was performed on PcArsR, spisPink, and AmilCP. We heat killed the ligase at 80ºC for 20 minutes the next day, and digested the new insert with XbaI and SpeI. After digestion, we did an overnight ligation of the vector and the insert using the slightly modified iGEM protocol with an overnight incubation.

## Testing ligation success before transformation

The team spent a lot of time transforming without successful insertion of the circuit, and needed a better way to determine the quality of ligation product that was being put into the cells. We reasoned that since PCR can amplify the tiniest amounts of DNA, we may be able to use this to screen the ligation products.
To screen the ligation, we ran end point PCR on the newly ligated parts using our standard PCR protocol. Surprisingly, the PCR confirmed that the vector and insert successfully ligated together! We then transformed our full circuit using the transformation protocol on the iGEM website, and mini-prepped using the Promega Wizard Plus SV Kit.

![](https://static.igem.org/mediawiki/2018/b/b6/T--Tacoma_RAINmakers--Week6LigationPCR.jpeg)

*Fig. 3. DNA gel of ligation showing successful ligation.*

# Week 9

## Bioengineering Summer Camp

The team took a week off from lab work to run a summer bioengineering camp for 9th and 10th grade students. We had a lot of fun teaching the students and used many tools gained from iGEM, like letting students streak out bacteria containing RFP (positive transformation control) and GFP (interlab positive control).

# Week 10

## Circuit testing

We started the process to test the complete arsenic circuit. In vivo testing of full circuits with reporters amilCP and spisPink was performed with 1uM/2uM/10uM concentrations of arsenate/arsenite.
RAIN's 2017 iGEM team had ran an experiment testing 150uM arsenic which killed the cells during the experiment. A second experiment was conducted where 25uM arsenic was used. This did not kill the cells and GFP was produced. This is the maximum amount of arsenic we want to test.

The limits of detection for our circuit to be viable and to compete with current testing equipment is below 100ppb. At 50ppb soil and water is considered unsafe for consumption or play. At 100ppb the soil or water is deemed worthy for excavation due to its potential health risks. Therefore, we will test 1uM arsenic (equivalent to 75ppb), 2uM arsenic (150ppb) and 10uM arsenic (750ppb).

10mL of liquid culture was created for both spisPink and amilCP the cultures will be set up as follows:
![](https://static.igem.org/mediawiki/2018/1/1b/T--Tacoma_RAINmakers--Week10-11TableArsenicTest.png)

The cultures were observed at 1 hour, 3 hours, 24 hours and 48 hours for visual solution color change. During the entirety of the experiment, no color change or production occurred.

## Repeat circuit testing with higher concentration of arsenite/arsenate

Testing of the amilCP and spisPink in vivo was unsuccessful at 1uM/2uM/10uM. There was also an attempt to grow CFU plates, however, these plates were left in the incubator for two days and overgrew. RAIN's 2017 iGEM team was able to see production of GFP at 25uM concentration of arsenic so this experiment will lie closer to these values.

Three concentrations or arsenic will be tested this time: 17.65uM (equivalent to 1324ppb), 21.42uM (1606ppb) and 25uM (1875ppb).
4mL of liquid culture was created for both spisPink and amilCP circuits.

After growing overnight, there were no color changes in the cultures.

# Week 11

## More cloning

Since we had a lot of trouble seeing any reporter expression, the team wanted to clone the individual parts of just the regulator and just the reporters. This will allow us to see if the reporter will turn on when its not being repressed with ArsR. Unfortunately, we ran into the same problems as before. By this, we mean that the ligation reactions were not demonstrating successful insertion of our DNA into the vector.

# Week 12-13

Different members of the team kept trying to clone the individual parts. The individual parts were amplified using our standard PCR protocol. Following PCR amplification, the reactions were run at 120V on a 1% Agarose gel. After seeing in the light box that the PCR was successful, the PCR products were extracted using a standard protocol and stored at -20°C.

![](https://static.igem.org/mediawiki/2018/6/6d/T--Tacoma_RAINmakers--Aug20AnnotatedGel.jpg)

*Fig. 4. DNA gel of PCR products.*

The purified parts and pSB1C3 were digested overnight with XbaI and SpeI.
The ligation was performed the next day using reactions contained 4.1μL of digested insert, 1.35μL digested linear pSB1C3, 1u T4 DNA ligase, and 1x T4 ligase buffer, and Molecular Water to a final volume of 10μL. It was incubated overnight at 16C.
Then, the ligation was tested with PCR primers amplifying the region between the XbaI and SpeI sites on pSB1C3. The PCR reactions contained 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.5μL of Ligation Product, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL. We ran it on a 1% agarose gel and saw no bands of the desired size.

In order to test the amilCP reporter, we created a circuit with a high expression T7 promoter, amilCP, and the same double terminator used in our other circuits. We began cloning by digesting IDT stock solutions of T7/amilCP with EcoRI-HF and PstI, following iGEM standard protocols. We then ligated T7/amilCP with pSB1C3, using the iGEM ligation protocol. To screen the ligation we used our PCR protocol. After running PCR products on an agarose gel, we found that the ligation failed.

# Week 14

We repeated the ligation at 4°C overnight. The reactions contained 5μL digested insert, 5μL digested linear pSB1C3, 1U T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 15μL. The ligation was tested in the same way, and once again produced no bands.

The third attempt at ligation contained 10μL digested insert, 2.5μL of a plasmid with pSB1C3 that we digested using XbaI and SpeI, 1u T4 DNA Ligase, 1x T4 Ligase Buffer, and molecular water to a final volume of 20μL. It was left at room temperature for 1 hour, then moved to incubate at 16°C overnight. The ligation was tested in the same way as the previous 2 attempts. The gel had strong bands at 75bp, the size of the empty vector, and faint bands in the correct location for successful ligations.

![](https://static.igem.org/mediawiki/2018/9/96/T--Tacoma_RAINmakers--Sept6AnnotatedGel.jpg)
*Fig. 5. PCR of successful ligation. Extremely faint bands are visible in the 500-1000bp range of Samples 2, 3, and 4!*

Transformations for the PArsR-spisPink construct were performed following the iGEM transformation protocol.
The transformations were screened with colony PCR reactions containing 1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL.

![](https://static.igem.org/mediawiki/2018/f/f3/T--Tacoma_RAINmakers--Sept7AnnotatedGel.jpg)
*Fig. 6. Colony PCR screening. Many colonies were screened to get just 2 positive colonies for the spisPink construct.*

In continuation of the amilCP test circuit, we went back to the stocks and digested T7/amilCP with EcoRI-HF and PstI, again following iGEM protocols. After digesting, we ligated the product, this time with a ratio of backbone to insert at 5:1, in an effort to make the reaction more efficient. We screened with our standard PCR protocol and found that the ligation was a success (fig. 7.).

![](https://static.igem.org/mediawiki/2018/1/1d/T--Tacoma_RAINmakers--MMLigationScreening.jpg)
*Fig. 7. T7/amilCP ligation screening. Sample 1 shows a band just under 1000bp, the correct size for ligated product.*

# Week 15

Now that one colony has been confirmed to contain the spisPink construct, we needed to transform the remaining ligations. Transformations followed the usual protocol, and colony screening by PCR.

With the ligation of the amilCP test circuit was complete, we proceeded to transform it according to iGEM protocols. Visual examination of the plates after incubation showed no signs of bacterial color change. We screened the colonies with our PCR protocol and found that the full circuit was present (fig. 8.). This likely means that there was a flaw in circuit design. Members of our team have speculated that this was due to the lack of spaces in the sequence around the promoters and ribosome binding sites.

![](https://static.igem.org/mediawiki/2018/a/ae/T--Tacoma_RAINmakers--MMTransformationScreening.jpg)
*Fig. 8. T7/amilCP transformation screening. Bands are visible just under 1000bp for Samples 2, 3, and 4. This corresponds to the size of T7/amilCP.*

# Week 16

After successfully transforming all our individual parts, we finally were getting ready to do minipreps for them. We used the Promega Wizard Plus SV Miniprep Kit and used the protocol provided by Promega to do our minipreps. The whole process did not take too long and soon after, we now had minipreps for pSB1C3/PcArsR, pSB1C3/PArsR-spisPink, and pSB1C3/PArsR-amilCP and stored them in the -20ºC freezer until it was time to send them to iGEM.

At this point, everyone on the team was back in school. Our subteam leaders were starting college and the rest of us were studying for SATs and preparing college applications. Now the team shifts focus to Jamboree!


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tacoma RAINmakers/Experiments

Team:TacomaRAINmakers/Notebook - 2017.igem.org

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:ECUST/Lab/Notebook

[![](https://static.igem.org/mediawiki/2018/3/3a/T--Tacoma_RAINmakers--teamlogo.png)](https://2018.igem.org/Team%3ATacoma_RAINmakers)

* [HOMEPAGE](https://2018.igem.org/Team%3ATacoma_RAINmakers)
* [Team](https://2018.igem.org/Team%3ATacoma_RAINmakers/Team)
  + [Sponsors](https://2018.igem.org/Team%3ATacoma_RAINmakers/Sponsors)
* [Project](https://2018.igem.org/Team%3ATacoma_RAINmakers/Description)
  + [Experiments](https://2018.igem.org/Team%3ATacoma_RAINmakers/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATacoma_RAINmakers/Notebook)
  + [Interlab](https://2018.igem.org/Team%3ATacoma_RAINmakers/InterLab)

  + [Attributions](https://2018.igem.org/Team%3ATacoma_RAINmakers/Attributions)
* [Parts](https://2018.igem.org/Team%3ATacoma_RAINmakers/Parts)
* [Safety](https://2018.igem.org/Team%3ATacoma_RAINmakers/Safety)
* [Human Practices](https://2018.igem.org/Team%3ATacoma_RAINmakers/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3ATacoma_RAINmakers/Public_Engagement)
* [Awards](https://2018.igem.org/Team%3ATacoma_RAINmakers/Awards)
* [Judging Form](https://igem.org/2018_Judging_Form?team=Tacoma_RAINmakers)

Team- 2018.igem.org

# Team:RAINmakers/Notebook

# Tacoma RAINmakers Experiments

# PCR Amplification

##

In order to begin the cloning process, individual portions (amilCP, spisPink, PcArsR) of our insert must be amplified to facilitate digestion and ligation of our plasmid. This PCR involves two different primer pairs; one pair matches to the chromoproteins and the other matches to the arsenic regulator.

The basic PCR master mix includes 1x GoTaq Flexi Green Buffer, 0.2mM dNTPs, 2mM MgCl2, 1.25U GoTaq Polymerase, 1μM FWD primer, 1μM REV primer, 1ng template DNA, and molecular water to a final volume of 25μL.

The typical thermocycler protocol heats the reaction at 95C for 5 min. Then it cycles through steps of denaturing (95C for 60s), annealing (60C for 10s), and extension (72C for 30-60s) for 35 cycles. There is a final extension of 72C for 5 min before the reaction is complete and held at 10C.

# Agarose Gel Electrophoresis

##

1. To prepare 1% agarose gel, measure 0.5g of agarose. Add to 50mL of 1X TAE buffer in flask. Swirl to combine.
2. Boil until liquid is homogenous and clear. Let cool. Add 5µL of Sybr Safe dye. Swirl gently until mixed.
3. Pour gel. Add desired comb. Let cool until translucent.
4. Add 1X TAE buffer solution into tank until gel is slightly covered.
5. Add purple loading dye to DNA sample, in necessary.
6. Add 10µL of ladder to your rightmost/leftmost well. The ladder is 1kb Plus Ladder from ThermoScientific (SM1333). Load 10-20µL of DNA per well.
7. Set for 120V for 15-30 minutes.
8. Image gel using blue light.

# Gel Extraction

##

1.After taking a picture of the results of the gel electrophoresis, use blue light along with an orange filter to help find the DNA bands.
2. Place a cut right underneath the band of DNA that is intended for extraction.
3. Cut a piece of filter paper and dialysis tubing to an appropriate size so that it would fit inside the slit.
4. Stick the tubing and filter paper together. Place this into the slit so that the side with the filter paper faces the band of DNA.
5. Place the gel in a gel box. Run the gel at 120V for 10 minutes or until the DNA moves into the filter paper and stays.
6. After running the gel, place the casting tray under blue light once again to ensure that the DNA has successfully moved onto the filter paper.
7. Take a 0.5mL centrifuge tube and pierce a small hole at the bottom of the tube. Place this small tube in a 1.5mL centrifuge tube.
8. Place the filter paper with the DNA into the small centrifuge tube. Centrifuge both tubes. This allows the supernatant to flow out of the small tube into the large one.
9. Take the small centrifuge tube out and discard it. The DNA has been extracted.

# Restriction Digest

##

1. Use SnapGene to determine which enzymes to use for digesting the desired DNA. Use NEB.com to determine which buffer is compatible with these enzymes.
2. Make master mix (1X Buffer, 1U enzyme A, 1U enzyme B, DNA, and molecular water to 25uL) in a centrifuge tube. Remember to keep the enzymes on ice and to add them to the master mix last.
3. Keep DNA on ice. Add DNA to the centrifuge tube with the master mix. Mix using the pipette.
4. Centrifuge the tube with the digestion to make sure any liquid on the sides of the tubes is at the bottom of the tube.
5. Place the digestion reaction in a water bath set at 37ºC for anywhere between an hour to overnight.

# Ligation

1. After the insert DNA and the vector backbone have been digested with the same enzymes, they can now be ligated.
2. The reactions contained a volume of digested insert, a volume of digested linear pSB1C3, 1uL T4 DNA ligase, and 1x T4 ligase buffer, and Molecular Water to a final volume of 10μL.
3. Incubate the ligation at 16°C overnight.

# Ligation Screening

##

1. Before transforming the ligation, we tested the ligation reaction using PCR to determine whether we could detect the presence of inserted DNA in the vector. This is a step that we have not seen many labs use and we were excited to implement it.
2. Using 1 uL of ligation reaction, perform PCR as usual using primers that target sites flanking the insertion site.
3. If the agarose gel shows a product of the correct size, proceed with transformation. If not, then re-ligate using a different proportion of Insert:Vector DNA and try again.

# Transformation

##

1. Label one 1.5 mL tube as a positive control (with vector and insert ligation) and one 1.5 mL as a negative control (vector only).
2. Thaw competent cells on ice. Pipette 50 uL of competent cells into both the positive and negative control tubes.
3. Pipette 2 uL of DNA into the competent cells. Gently mix by flicking the tubes.
4. Incubate both tubes on ice for 30 minutes.
5. Heat shock the tubes at 42ºC for 45 seconds. Put the tubes back on ice for 2 minutes.
6. Pipette 900 uL SOC media to the positive and negative control tubes. Be careful not to contaminate the SOC.
7. Place the tubes in a shaking incubator. Incubate at 37ºC for 1 hour at, shaking at 200 rpm. About 15 minutes before incubation finishes, remove plates from the fridge, label appropriately and leave them at room temperature.
8. Take tubes out of incubator. Spin cells at 6000 rpm for 30-60 seconds.
9. Pour off the supernatant, leaving 100-200 uL in each tube.
10. Gently resuspend the pellet in the tubes using the supernatant left.
11. Pipette the resuspended cells onto the selection plate.
12. Add 15-20 glass beads to each plate. Put lid back on each plate. Shake both plates so that the cells can spread around. Once done shaking, pour the beads out and dispose of them properly.
13. Incubate both plates upside down at 37ºC for 14-18 hours.

# Colony PCR Screening

##

The goal of this PCR is to take single colonies from transformation plates and to amplify the DNA in the positive control (ligated vector and insert) and negative control (vector only). After screening, gel electrophoresis will occur to determine whether or not the vector and insert ligated to each other successfully (do this by checking the base pair count).
1. After running a transformation, prepare a master mix (1x GoTaq Flexi Green Buffer, 0.15mM DNTP, 1.5mM MgCl2, 0.63u GoTaq Polymerase, 0.38μM FWD primer, 0.38μM REV primer, and molecular water to a final volume of 10μL). Add an extra 20% uncertainty to each volume of reagent to ensure there is enough mastermix for all PCR tubes.
2. Make sure master mix is mixed using pipette. Then, put 20 uL of the master mix into each PCR tube.
3. Take out the transformation plates from the refrigerator. Also get a new LB-CAM plate (will be used to streak a single colony onto new plate).
4. Select a colony, place a toothpick on it, and dab the same toothpick onto the new petri plate. Then use the same toothpick to inoculate a PCR reaction. Finish streaking the plate using a round-ended toothpick. Properly discard both toothpicks. Repeat until completed for all PCR tubes.
5. Centrifuge the PCR tubes. Then, set the thermocycler so that it has the correct settings (since each PCR reaction is different. Place the PCR tubes in a thermocycler, and start the reaction.
6. Incubate the new petri plate (with the streaks) upside down at 37ºC for 14-18 hours.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tacoma RAINmakers/Results

Team:TacomaRAINmakers/Notebook - 2017.igem.org

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:ECUST/Lab/Notebook

[![](https://static.igem.org/mediawiki/2018/3/3a/T--Tacoma_RAINmakers--teamlogo.png)](https://2018.igem.org/Team%3ATacoma_RAINmakers)

* [HOMEPAGE](https://2018.igem.org/Team%3ATacoma_RAINmakers)
* [Team](https://2018.igem.org/Team%3ATacoma_RAINmakers/Team)
  + [Sponsors](https://2018.igem.org/Team%3ATacoma_RAINmakers/Sponsors)
* [Project](https://2018.igem.org/Team%3ATacoma_RAINmakers/Description)
  + [Experiments](https://2018.igem.org/Team%3ATacoma_RAINmakers/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATacoma_RAINmakers/Notebook)
  + [Interlab](https://2018.igem.org/Team%3ATacoma_RAINmakers/InterLab)

  + [Attributions](https://2018.igem.org/Team%3ATacoma_RAINmakers/Attributions)
* [Parts](https://2018.igem.org/Team%3ATacoma_RAINmakers/Parts)
* [Safety](https://2018.igem.org/Team%3ATacoma_RAINmakers/Safety)
* [Human Practices](https://2018.igem.org/Team%3ATacoma_RAINmakers/Human_Practices)
  + [Public Engagement](https://2018.igem.org/Team%3ATacoma_RAINmakers/Public_Engagement)
* [Awards](https://2018.igem.org/Team%3ATacoma_RAINmakers/Awards)
* [Judging Form](https://igem.org/2018_Judging_Form?team=Tacoma_RAINmakers)

Team- 2018.igem.org

Loading menubar.....

# Team:RAINmakers/Notebook

# Tacoma RAINmakers Results

# The Results

---

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.