sTAUbility

Lab Notebook

We have conducted two experiments: the "Gene-SEQ" experiment (Measurement, large-scale of a Co-culture), and the "10-genes" experiment (POC, small-scale). For more information, visit our Experiments page.

The protocols we refer to in this notebook appear in our Protocols page.

Tuesday 28/01/2020 - 10 gene and Gene-SEQ

We handled the yeast strain library that we received from Professor Maya Schuldiner's lab as written in the “Handling+Freezing the engineered yeast library - first time protocol”. The library contains 2 copies - GFP library + RFP library, each separated into 6 plates. Every strain position is explained according to the excel document “SWAT library”.
In addition, the Gene-SEQ experiment started according to the “Starting the evolution experiment - Gene-SEQ” protocol. 96 Well Plate first Legend below.
Wednesday 29/01/2020 - 10 gene and Gene-SEQ

We have conducted 2 fluorescence tests to analyze the strain's growth in the previous day.
The first fluorescence reading was conducted using a fluorescence black plate 96-wells as shown in figure 1. Each well contains 100µL of the matching strain.
The results of this test are summarized in folder “Results: Fluorescence Top Reading and Absorbance” and are divided into 3 sections:
1. Green Fluorescence Top Reading (label 1)
2. Red Fluorescence Top Reading (label 2)
3. Absorbance\OD (label 3).
The results indicated that the YPD medium disrupted the measurements, because of its natural fluorescence. Therefore we have decided to conduct another experiment and to dilute the strains with “SD” and “-Ura” solution .

The second fluorescence reading was conducted using a fluorescence black plate 96-wells as shown in figure 2. Each well contains 1:10 dilution of the strains with SD and -Ura: We’ve transferred 50µL of the strain solution to a 200µL SD solution, and then transferred 50µL of the outcome solution to a 200µL -Ura solution. The results of this test are summarized in folder “Results: Fluorescence Top Reading and Absorbance” and are divided into 3 sections as mentioned above.
1. Green Fluorescence Top Reading (label 1)
2. Red Fluorescence Top Reading (label 2)
3. Absorbance\OD (label 3).
The results indicated that the use of SD lowered the noisy signals achieved from the YPD natural fluorescence.

Therefore we prepared SD complete solution (see media preparation) to be our new growing media and continued according to protocol with a new organization for the plates, in which all the empty wells is where the second experiment should be placed.
Thursday 30/01/2020 - 10 gene and Gene-SEQ

We followed the protocol “Updated Gene-SEQ protocol with SD media”. Results excel files were added to the results folder.
While observing the fluorescence results, we noticed that the absorbance (OD) of plate “1” cells was lower than expected, compared to the test plate “2” OD: Approximately 0.2 average OD higher than plate “2” (multiplying the diluted cells OD in plate “1” by 5). It should be higher than that, considering the cells grow in plate “1” and not in plate “2”.
We assumed that this difference in expectations might be because we use different plates at the absorbance/fluorescence tests. Plate “2” was tested in a transparent 96 well plate, and plate “1” was tested in a black 96 well plate.

Therefore, for the next test we do (sunday - 2/2) we will use 96 black plate for both tests.
Friday 31/01/2020 - 10 gene and Gene-SEQ

We detected a murky solution in column 12 of the infection plate (-leu) that may indicate the growth of infection. This may have been due to a mistake of the previous day- insertion of a little -ura substrate into the -leu substrate wells.
We marked the plate and returned it to the incubator for a deeper test the following day and continued the experiment according to the protocol.
We noticed that the experimental protocol could be improved by saving a 96 well plate and improving infection prevention. We updated the protocol and sent for approval of the lab manager .

Saturday 01/02/2020 - 10 gene and Gene-SEQ

Probably as a result of using the wrong media for the contamination plate, there was a considerable growth in this plate, we prepared a new contamination plate and the results tomorrow will indicate the reason for the growth, if because of wrong media or because a contamination.
- We found a mistake in the genetic background of the negative control for the RFP library and therefore changed it for a different one from the RFP library.
- We added a WT as a negative control to the plate. This control should grow in the contamination plate as a positive control and should not be fluorescent in addition to serve as a baseline of the natural fluorescence of yeast.
Sunday 02/02/2020 - 10 gene and Gene-SEQ

We did everything as written in the protocol. There was not anything special in the results. We prepared new Glycerol plates and froze them as needed on Sunday.
Monday 03/02/2020 - Gene-SEQ

We followed the “Updated Gene-SEQ protocol with SD media”
.
There was no remarkable growth on the contamination plate except for the control well. The results have been uploaded to the ‘Results’ folder.
Tuesday 04/02/2020 - 10 gene and Gene-SEQ

The protocol was followed with no apparent problem.

One of the pipettes in the multi pipette is not working properly, so whoever uses it should make sure that the liquids rise enough.

When doing the OD test, we realized that the WT in H11 grew but not as much as expected, this could have been because the solution was not well mixed when collecting the sample, the day prior.

Besides that we saw that in the results of the day before (3/2) A8 had an OD of 0.6 in comparison to an average of 0.1, on the other hand, the results today were normal and didn’t show signs of contamination.
Wednesday 05/02/2020 - 10 gene and Gene-SEQ

The protocol was followed with no apparent problem.

In the contamination plate, in 10B there was growth, we continued as usual and tomorrow based on the growth in the contamination plate, we will decide if the contamination was only in the contamination plate or also in the main plate and will behave accordingly.
Thursday 06/02/2020 - 10 gene and Gene-SEQ

In the contamination plate, in 10B there was growth, we replaced 10B with 9B and we left 9B as is.

From the OD test we noticed that the WT in H11 grew but not as much as expected around (0.19). In the dilution plate in line B rows 7-12 the liquid was higher than the rest, so we filled H rows 1-6 instead.
Friday 07/02/2020 - 10 gene and Gene-SEQ

We conducted the experiment as usual and no contamination has been observed. In the WT control in plate 2 growth was smaller than usual.
Saturday 08/02/2020 - 10 gene

We conducted the experiment as usual and no contamination has been observed.

EMS Calibration:

In order to evaluate the correct concentration of EMS for our experiment, we constructed a calibration protocol. (see EMS Calibration protocol) EMS calibration link
Sunday 09/02/2020 - 10 gene and Gene-SEQ

There was a clear contamination in at-least four wells. We defrosted the plate from the 3/2/20 and continued according to the protocol.
Monday 10/02/2020 - 10 gene and Gene-SEQ

All plates were apparently contaminated (including samples from the fridge/freezer). We aborted the experiment in order to have a fresh start of the full experiment, including:
1. Gene-SEQ Experiment.
2. Individual strains experiment.
3. EMS experiment.
Tuesday 11/02/2020 - 10 gene

EMS Calibration results:

5μl of EMS in 500ml is enough to increase the mutation rate in 100-fold while causing 20% lethality or decrease in growth (as calculated from OD compared to a control that was not given EMS).

OD of cells:
- No ems -1.34
- 1μl ems - 1.1256
- 5μl ems-1.072
- 10μl ems- 0.93
- 25μl - No growth
- 50 μl - no growth
Mutation as measures on CAN plates:
- No ems -8
- 1μl ems - 379
- 5μl ems-852
- 10μl ems- 531
- 25μl - 0
- 50μl - 0
We started the full experiment according to the “Evolution experiment” protocol.

The strains selected by the modeling team are:

TEL2, SEC21, CEG1, KAP95, PMI40, GCD10, HCA4, CPC9, SQT1, TOL3.

GFP positive control - Ura+ - 1-27-41. (WT)

RFP positive control - his3d GFPdc - 1-5-40.

All from the SWAT GFP/RFP library.
Wednesday 12/02/2020 - 10 gene and Gene-SEQ

We continued the full experiment according to the protocol and initiated the first contamination plate.

Both "result" files (for plate “1” and “2”) contain the O.D, fluorescence and absorbance measurements.
Thursday 13/02/2020 - 10 gene and Gene-SEQ

Evolution experiment aborted due to a problem in the Gene-SEQ (including positive control strains) and model changes, we realized we required different strains for the 10 individual strains experiment.

Thursday 05/03/2020 - 10 gene

Evolution Experiment was re-initiated with 10 new individual strains chosen by our model team: According to “Evolution Experiment - Initiation Protocol”

EMS experiment - WAS NOT STARTED .
Friday 06/03/2020 - 10 gene and Gene-SEQ

In the past few days, we did not use a flame next to our plates and materials, because of a university shortage in gas tanks that are imported from china. Contaminations may arise.

We initiated the EMS experiment (Initiation protocol).
Saturday 07/03/2020 - 10 gene and Gene-SEQ

Contamination visually observed in most of the Gene-SEQ GFP negative controls. In addition, no growth detected in the WT control. We suspect that contamination developed during storage of the libraries. Moreover, in plate 2-EMS, very little growth was detected. Due to these problems, we decided to continue the experiment until tomorrow and see whether there is contaminations/growth with proper examinations.

Correspondingly to observations, we will decide whether to apply to Schuldiner’s lab for new libraries and may change our protocol to minimize contaminations in the future.
Sunday 08/03/2020 - Gene-SEQ

All the Gene-SEQ-GFP was contaminated (Row A). We put instead of the GFP more negative control of RFP in the new plates. Matan prepared new SD Complete. In plates 3+4, in row 1, columns 1-10 we put SD-URA.
Monday 09/03/2020 - 10 gene and Gene-SEQ

We decided to let the EMS plates (2+4) incubate another day because there was not any visual growth in the wells.

We started the daily protocol, and while transferring from plate “2” (Contamination control) to the OD plate, we visualized turbidity in most of the wells in the first row, last column and a few more turbid wells in the individual genes wells (GFP and RFP). All of which are negative control for growth and therefore all contaminated.

We decided to abort the experiment (Excluding EMS), restart fresh, and figure out a way to make this experiment cleaner with less contaminants.

Tuesday 19/5/2020 - 10 gene(link missing)

We had 2 goals:
1. validating the 10 chosen genes for our evolution experiment.
2. Creating a negative control for the GFP and for the RFP for the evolution experiment.
For the first, we used the correlated species we isolated before from Shuldiner yeast library. Then we followed the protocol for DNA extraction. We designed primers for each of the 10 genes and we used them with the PCR protocol we followed.

For the second goal we used primers we designed earlier (CNG - for the GFP, CNR- for the RFP). We needed to replace the Can1 gene with a GFP and RFP accordingly. In addition we inserted a stop codon to the primer, designed to enter after the GFP and the RFP, so that we will get the fluorescent protein (the fluorescent level was extremely low because the GFP/RFP are fused to the Can 1 gene). We followed the DNA extraction protocol and the PCR protocol.

Later we tested the PCR products by gel electrophoresis, here are the results:

results link.
Thursday 21/5/2020 - 10 gene missing link to table

We performed a second PCR to validate the 10 genes and create the negative control amplicons. We have changed the elongation temperature for each gene according to the results we got in the first PCR (see the table in day 25/5/20).

In addition, we tried to transform the negative control for the CAN1 species. We have done successfully with the GFP, the RFP did not work. We decided to re-design primers for the RFP.

We tested the PCR products in gel, here are the results:

We documented the results in our excel table (link).
Monday 25/5/2020 - 10 gene

We performed a third PCR for validating the 10 genes and creating the negative control amplicons. We have changed the elongation temperature for each gene according to the results we got in the second PCR (see the table below). Here are the results(documented in our excel table) :

We decided to re-design primers for the genes we have not managed to validate.

Tuesday 2/6/2020 - 10 gene

Because we did not get a product after 3 PCR runs of CDC9 strain for GFP, we decided to re-seed the strain onto an agar plate and re-extract DNA according to the attached protocol. Then we froze the DNA products.
Tuesday 9/6/2020 - 10 gene

After we re-extracted the DNA from the CDC9 strain, we ran a PCR and got no product at all. We assume that this strain is not CDC9. in order to validate this we ordered different primers that will give a different PCR product in different cases:
1. If it is the CDC9 strain, we get a 4KB PCR product containing the GFP fused to CDC9 gene
2. If it isn’t the CDC9 strain, we get a 1KB PCR product containing the GFP only.
Update: we performed the PCR and got 1KB product. We successfully confirmed that this specific strain is not CDC9. Unfortunately we can not know which strain it is at this stage.
Wednesday 10/6/2020 - 10 gene

We performed calibrations for OD measurements and GFP measurements of our plate reader. We used the iGEM official measurement protocols (with measurement kit from previous year). https://2020.igem.org/Measurement/Protocols#kit_protocols We will order an RFP calibration material for future experiments that we intend to do with mcherry protein.
Wednesday 10/6/2020 - 10 gene

We performed another PCR for the negative control of our 10 genes experiment, using the new primers we designed. Unfortunately we still didn’t get any PCR product. Again, we decided to redesign new primers.
Monday 15/6/2020 - 10 gene

We seeded the ten strains (of the 10 gene experiment, RFP/GFP) from our frozen stock to -ura plate (GFP) and not+ plate (RFP) , in order to have fluorescent cells available for FACS analysis. The goal is to eventually make a FACS analysis of the Gene-SEQ frozen stock, and a FACS separation of fluorescent cells for further experiments.
Thursday 18/6/2020 - 10 gene

We took the ten strains of GFP that were seeded and started the evolution experiment on those strains. This time the experiment will be strictly inside a biological hood to further prevent any contamination. We also initiated the RFP strains although we still do not have a negative control for it.

first FACS analysis of 10 genes (GFP/RFP) were done. Details added later.
Friday and Saturday 19-20/6/2020 - 10 gene

Contaminations arose in some of the wells of the RFP strains. So, the experiment was stopped, we decided to do an isolation seeding to those strains again in order to get rid of the contaminations.
Sunday 21/6/2020 - 10 gene

We did not detect any growth in the wells of the WT, so we decided to take new colonies for the WT control as it is only used as a measurement base control for the fluorescent emission. No contaminations whatsoever.
Monday 22/6/2020 - 10 gene and Gene-SEQ

We noticed that the measurement fluorescence protocol of Schuldiner's Lab is not suitable for the plate reader we have, so the results were not good enough. We changed the protocol and stopped the experiment because of technical reasons.
Thursday 25/6/2020 - Gene-SEQ

First FACS sorting - mixed cultures. Results will be shown later.

Friday 10/7/2020 - 10 gene

The 10 genes experiment was started today with the new protocol for fluorescence.
Saturday until Wednesday 11-15/7/2020 - 10 gene

The experiment went smooth with no contamination. The experiment was stopped once again due to technical issues. We froze the strains and will continue the experiment on Sunday.
Monday 13/7/2020- 10 gene

Starters for tomorrow’s FACS were made.

After isolating all the 10 genes from frozen stock we took a picture of them with fluorescent microscopy, they were all fluorescent (at different levels).

RFP

GFP
Tuesday 14/7/2020 - Gene-SEQ

For the first time we have made a FACS-sort to our Gene-SEQ (GFP/RFP). The goal was to separate contaminations or unwanted cells from our GFP/RFP- expressing strains in the Gene-SEQ, by fluorescence emission differences. Results files were saved in the drive.
- Tube 1 - BY control, for calibration.
- Tube 2 - a mixed culture of TSC13-GFP strain and BY control, in order to examine a simple separation of two strains with high differences in fluorescence. (TSC13 was found to have highest level of GFP fluorescence emission from all 10 genes).
- Tube 3 - Gene-SEQ GFP
- Tube 4 - Gene-SEQ RFP
After sorting, tubes 2-4 were centrifuged and separated from PBS buffer(from FACS machine) and transferred to the following media for growth: T2 - SD-complete. T3/T4 - one tube of SD(-leu) and another tube of SD(-ura).
Thursday 16/7/2020 - Gene-SEQ

Unfortunately, some of the tubes from the FACS sort were lost after being placed in a lab’s incubator. The sorting of the Gene-SEQ must be done again.
Sunday 26/07/2020 - 10 gene

We followed the “new protocol 10 genes experiment July 2020” for plate 1 and plate 3, including a fluorescence measurement.

We also performed an OD check for yesterday’s strains.

The results were saved in the “10 genes fluorescence daily results” folder.
Monday 27/07/2020 - 10 gene

Unfortunately, contaminations occurred on all of the wells, so the experiment was stopped once again and started over from a frozen stock of day 5.

From now on only one person will conduct the experiment to decrease the contaminations to the minimum.
Monday 27/07/2020 - 10 gene

We performed a final PCR for the 10 different strains we are using on the “GFP 10 genes experiment”. Except for one strain we already knew was not the expected (CDC9), we have successfully validated all the GFP strains, using electrophoresis. We documented the results in our “Results” wiki page.

We designed primers for Sanger sequencing to each one of the strains.
Monday 27/07/2020 - Gene-SEQ

A FACS sort was made in order to clean the co culture (from frozen stock) by its GFP/RFP fluorescence. After 2 days the RFP negative control showed growth, and after 3 days the GFP negative control showed growth as well. The experiment was terminated.

28/07-9/08/2020 - 10 gene

The experiment continued smoothly without contaminations so far. the measurements were uploaded to the results files in the drive.
Monday 10/8/2020 - Co cultures

In the previous day we have made starters of GFP/RFP co cultures, the GFP in SD-URA and RFP in SD-complete + NAT (1:1000). That might be the reason previous experiments went wrong.

The RFP culture has a NAT resistance selection, which was not used before. The major mistake was growing the RFP culture in -Leu media. After reviewing articles about those yeast strains, the BY4741 strain (which is used as control and as basis of the libraries).

The libraries have a +Ura strain which should grow on -Ura media, this should affect the results, meaning that when RFP culture is transferred to -Ura media as negative control, the +Ura strain can grow!.
10/8-16/8/2020 - 10 gene

Up until now the experiment went well. Today a contamination occurred in all the wells.
After investigation we concluded the plate itself was not washed thoroughly. Unfortunately, we had to start the experiment again from a frozen stock of day 10.
13/8/2020 - 10 gene

We want to send samples from the 10 gene experiment for sequencing, so today we started preparing them, we did DNA extraction, PCR and clean up.
Sunday 17-23/8/2020 - 10 gene

The experiment was going as should be with some changes. From now on, the plates will be washed with water thoroughly until no residue is observed and soaked in bleach overnight, then put to dry until the next cycle of autoclave, in addition, the breathable stickers will be changed every day. For a final precaution, a batch of each strain will be stock in the refrigerator everyday in case of contaminations on the next day.
Monday 31/8/2020 - Gene-SEQ:

Apparently, Matan informed today that our frozen stock of the Gene-SEQ contains two distinct copies, one of them is the “Old” one, which is the first one we made with all the controls. We have used this one for all experiments until this day. The other one is the “New” one which is a copy that from where Matan tried to remove some of the controls, this stock might be contaminated according to Matan.

We have made starters for GFP/RFP co cultures each from the New/Old, 2 repetitions for each one, total of 8 tubes (RFP- comp+nat media | GFP- -Ura media). In addition, we have made negative controls for each GFP/RFP/New/Old - Total of 4 controls. Contained -Leu(old)/-His(new) media - The cells should not grow.

Tuesday 1/9/2020 - Gene-SEQ

There was some growth in the -His negative controls (New Gene-SEQ). We assumed that some negative controls (with the ability to synthesize Histidine) remained in the New culture and decided to use it anyway, assuming that the controls were not harmful for the results.

Started the Chi-Bio Gene-SEQ experiment. 2 vials of each GFP/RFP New Gene-SEQ. We added a small amount of cells to start the experiment. The machine is connected to a 1 liter bottle of media
Wednesday 2/9/2020 - 10 gene

We prepared 8 of 10 genes for sequencing. The genes were divided into 4 groups:

1. GFP with reverse primer.

2. GFP with forward primer.

3. RFP with reverse primer.

4. RFP with forward primer.

The prepared samples were frozen.
Thursday 3/9/2020 - 10 gene

The preparation for sequencing was successful on almost all the genes, so we sent them for sequencing and prepared again the ones that didn't succeed in addition to the 2 that were not prepared yesterday (the initial primers we use for the 2 genes did not work properly).
Thursday 3/9/2020 - 10 gene

In order to get the best results out of the plate reader, our instructor Dan advised us to precipitate the yeasts and change their solution to PBS before checking the fluorescence levels.
Thursday 3/9/2020 - 10 gene

In order to get the best results out of the plate reader, our instructor Dan advised us to precipitate the yeasts and change their solution to PBS before checking the fluorescence levels.
Monday 7/8/2020 - Gene-SEQ

Doron connected a 4 liter bottle of SD complete media to the Chi-Bio, should last for 3-4 days.
Tuesday 8/9/2020 - Gene-SEQ

One of the GFP vials might be contaminated, the machine wasted 4 liters of media (prepared yesterday) trying to resolve the high OD. Another explanation might be that cells were stuck to glass vial and interrupted the OD reading, thus maintaining a false and a very high OD read.

Both the GFP (contaminated) and RFP (clean) remain at the vials without media. Tomorrow we will examine the GFP for contamination and connect a new media bottle for the 2 RFP vials.
Sunday 9/9/2020 - Gene-SEQ

We found out that the negative control for the RFP library (RFP not fused to any gene) is not fluorescent and as such should not be used in the experiment and we decided to clone him from the original. To do this we ordered primers that attach on both sides of the RFP construct (with the selection marker and the promoter) and added in the primers homology to the locus we want to insert the construct, and because we took the construct from a strain in which the RFP is fused to a gene, we added a stop codon to the RFP. we run the PCR, got the band in the size that we expected.

The primers used:
- F- AGATAAGTAGATAAGAGAATGATACGAGATAAAGCACAAAcgtacgctgcaggtcgacggtggcggttctggcggtggcggatcc
- R- TACATTAGTATTAGCGTGAAAATGTACACATATACATATAaggaaccTTActtgtacagctcgtccatgc
In capital letters - created homology, in bold, mutation (inserted stop codon)

We did a transformation to yeast according to the protocol described in the protocols section, but for some reason did not get any colonies (while getting colonies in the positive control.) because of time consideration and the corona lockdown we decided to not try again.
Monday 21/9/2020 - Gene-SEQ

Because there are no suitable optional primers for deep sequencing of the GFP/RFP library, if we want to also sequence the ORF the SWAT marker is attached to, we had to find a better solution. We decided to cut the DNA with restriction enzymes that the engineering team found for us. That were present in all the SWAT library (fluorescent protein and its promoter and contained enough of the ORF in order to identify it.), then we will ligate the fragment to create a circular DNA fragment and by using primers from L2 linker we can now PCR all the fragments and send them to sequencing.

Afterward we learned that in order to achieve good sequencing we need to ligate the PCR fragment so we will be able to do a RCA reaction on it (rolling circle amplification), in this manner we will get a concatemer with many repeats of the PCR fragment, and in such manner we can distinguish between mutation and sequencing errors.

Restriction enzyme illustration:

Ligation illustration

PCR amplification illustration

Table showing the amount of DNA achieved per microliter after DNA extraction from yeast in nano grams (protocol in protocols ) after checking in nano-drop.

Then we used the resection protocol on all the samples and left for overnight in 37 degrees Celsius. We also conducted the same protocol on a control of one of the 10 genes (sec2), so we can check if the protocol was successful.
Tuesday 22/9/2020 - Gene-SEQ

We finished the reaction by incubating in 65 degrees for 20 minutes. Then we performed overnight ligation protocol as stated in protocols.
Wednesday 23/9/2020 - Gene-SEQ

We stopped the ligation reaction by incubating in 65 degrees for 20 minutes and then we used all volume from the ligation protocol for a PCR reaction. We first performed the PCR on the control, after validating that we got the expected band size (2.4kb)

We decided to move on to the rest of the DNA samples. Because the band was weak, we decided to increase the number of cycles to 50 (instead of 40). Afterward we validated the control by further sending it for sequencing, and came back with 300bp inside the GFP in very high assurance level (2e-58). We then did the PCR reaction on the rest of the sample, cleaned the PCR product, checked the amount of DNA in nanogram again in the nanodrop machine and did a ligation protocol as stated in the protocol. Added a table with the amount of DNA in nanogram per microliter in each sample.
Wednesday 23/9/2020 - 10 gene

We extracted DNA, did PCR and cleaning from the latest samples on the experiment until today, to send for sequencing.

this will most probably be the latest results we will get until the competition.
Thursday 24/9/2020 - Gene-SEQ

We stopped the reaction as stated in the protocol and send the DNA the Netherlands for sequencing at Cyclomics(Alessio Marcozzi PH.D is a Co-founder and former IGEM competitor ).
Thursday 24/9/2020 - 10 gene

We perform another PCR for the RFP negative control of our 10 genes experiment, using the new primers we designed. Unfortunately we still didn’t get any PCR product. We decided to postpone the RFP 10 genes experiment and continue only with the GFP.
Friday 25/9/2020 - 10 gene

We followed the sanger sequencing protocol and performed it on the 10 strains of our experiment (except CDC9 strain). Those are the strains of day 0 of the experiment.

Update: The sequencing results successfully validate the 10 strains (except CDC9) - in each one of them we got GFP fused to the gene we have expected.

Sunday 04/10/2020 - 10 gene

We created starters for each strain, using samples we took from day 26 of the experiment.
Monday 05/10/2020 - 10 gene

We performed DNA extraction for each of the strains in day 26 of the experiment (except CDC9). We then confirmed the extraction by PCR followed by electrophoresis. We documented the results in our “results” wiki Page.
Tuesday 06/10/2020 - 10 gene

We followed Promega Protocol and performed a PCR clean-up to the PCR products we got yesterday. We then followed the Sanger sequencing protocol and sent the samples to the sequencing lab.

Team:TAU Israel/Notebook