Team:UCopenhagen/Notebook/Week 29







Week 29 (15th-21st of July)

15th July 2019

1. Colony PCR of transformed yeast

Team members: Morning: Signe and Mads, Afternoon: Selma and Jonas

The colony PCR was done on the following transformed yeast cells:

Sample no. Fragments
1 GPER+G𝛂s
2 GPER-G𝛂i
3 GPER-Li-sfGFP
4 XLHCGR+G𝛂s
5 XLHCGR+G𝛂i
6 XLHCGR-Li-sfGFP

Materials

  • NaOH solution
  • MangoMix master mix solution
  • F1-primer
  • F2-primer
  • F2-primer
  • R1-primer
  • Template: Colonies from overnight plates
Materials Quantity (μL)
20 mM NaOH 20
2x MangoMix (commercial mastermix solution) 5
F1-primer 0.5
F2-primer 0.5
R1-primer 0.5
milli-Q-water 3.5
In total 10

Table 2: The composition of materials to be used per colony PCR amplification sample

Procedure
  1. Colonies were marked on the plates by circle and numbered.

    4 colonies were marked from the plates with the 3 assembly system (the samples with sfGFP - sample no. 3 and 6)

    8 colonies were marked from the plates with the 5 assembly system (the rest of the samples)

  2. Lysing of the yeast cells:
    1. For each colony a PCR tube was made ready by adding 20 µL of 20 mM NaOH to it.
    2. The colonies were taken from the plate by using a pipette tip.
    3. The colonies were inoculated by dipping the tip in the NaOH.
    4. The samples were spun down and then boiled by using PCR machine 1 and the “iGEM Dirtb” program. This program boils the cells for 10 minutes at 99 degree celsius
  3. Working solution of primers:
    1. For the colony PCR three primers are needed: YEA90 (F1-primer), YEA85 (F2-primer) and YEA89 (R1 primer). For more information about this look in the lab book.
    2. The working solution were made by doing a 1:10 dilution of the primers. For each primer 10 µL primer and 90 µL milli-Q-water was added to an eppendorf tube.
  4. PCR reaction:
    1. We use a commercial mastermix called MangoMix. This mastermix solution contains taq polymerase and dNTPs and therefore this does not need to be added as you would normally do.
    2. Mastermix - for each sample the following was added: 5 µL of MangoMix , 0.5 µL of each of the three primers (YEA90, YEA85 and YEA89) and 3.5 µL of milli-Q-water to a total volume of 10 µL.
    3. 9,5 µL of the Mastermix were added to the PCR tubes for all the samples.
    4. 0,5 µL of the corresponding templates were added to the PCR tubes (the PCR tubes are labeled 1-6 corresponding to the templates). The templates were spun down before so the 0,5 µL should be taken from the top, and not the bottom containing a pellet with cell debris.
    5. The tubes with the mastermix and the templates were spun down.
    6. They were put into the PCR machine #1, and the program “Igem Yeast” was used.
  5. Electrophoresis:
    1. A gel was casted.
    2. The samples were loaded on the gel.

Igem Yeast:

32 cycles of tex marked in red.

  • 96 °C: 30 seconds
  • 96 °C: 30 seconds
  • 55 °C: 25 seconds
  • 72 °C: 2 minutes and 30 seconds
  • 72 °C: 5 minutes
  • 12 °C: Infinity
Results

Discussion

No positive bands where found in the gel. We discussed with Lukas why this did not work out and decided to redo the yeast colony PCR tomorrow.

16th July 2019

1. REDO OF YEAST COLONY PCR

Team members: Jonas and Iben (Hitesh)

We discussed yesterday’s results with Lukas. Since there were no positive bands in the gel, he suggested that the quality of our DNA fragments might not have been great enough for successful transformation. He proposed that we redo the yeast colony PCR for the GPER-sfGFP yeast strain, since these DNA fragments were of high quality. Since only 4 colonies were picked for PCR, we might just have been unlucky, picking 4 negative colonies. Therefore, Lukas suggested we picked 24 colonies this time, to increase our chances of finding positive colonies.

Materials
  1. Materials and methods were the same as the previous yeast colony PCR
  2. 24 colonies were carefully picked from the plate
  3. PCR runs were increased from 32 → 35
  4. Positive and negative controls were added
  5. Samples were loaded in the gel 1 → 24, pos., neg.
Results

2. O/N Cultures for glycerol stocks

Team members: Selma and Iben

Preparation of overnight cultures to make glycerol stocks.

Materials
  • LB medium
  • Carbenicillin

    O/N cultures were prepared from the following E. coli strains:

  • GPER
  • GPER-lin-sfGFP
  • XLHCGR
  • XLHCGR-lin-GFP
  • STE12
  • Gαi
  • ZsGreen

All strains were inoculated into a tube containing 10 ml LB medium + 20 µl carbenicillin.

The tubes were incubated overnight at 220 rpm at 37°C.

17th July 2019

1. Glycerol stocksConstruct and primer design

Team members: Iben and Signe.

Glycerol stocks were made for the following fragments: GPER, GPER-sfGFP, XLHCGR, XLHCGR-sfGFP, STE12, Zs Green and G𝛂i.

Materials
  • 50% glycerol
  • Overnight cultures
  • Cryotubes
Procedure
  1. Add 500 µL 50% glycerol to each of the cryotubes (one cryotube to one sample of fragment)
  2. Add 500 µL of each of the O/N cultures to the respective tubes.
  3. Put the cryotubes in the -80 degree Celsius freezer.

2. Plasmid purification

Team members: Iben and Signe

We wanted to extract the DNA from the following samples: GPER, GPER-sfGFP, XLHCGR, XLHCGR-sfGFP, STE12, Zs Green and G𝛂i (G𝛂s was not extracted as we could not find the stock. This need to be done another time).

Materials
  • E.N.Z.A DNA mini prep kit

Procedure

  1. The protocol given in the E.N.Z.A DNA mini prep kit was followed.
  2. The protocol was changed as we took a bigger volume of culture (10 mL instead of 5 mL). As the volume was doubled the all solution volume was doubled as well.
  3. We also used the vacuum sucker from Karel’s lab to suck out the filtrate from step 8 onwards which is supposedly a better option than centrifugation.
  4. The binding column was taken out of the vacuum sucker and then put onto the microcentrifuge tube to elute the sample.
  5. Elution was done with 100 μL elution buffer which was heated to 60 degrees before pouring into the binding column and the buffer was left to sit on the binding column for about half an hour and then centrifuged.
  6. Nanodrop was afterwards done to measure the DNA concentration of the samples and these changes in the protocol yielded really great concentrations of purified plasmids.
Data
Sample Concentration (ng/μl)
GPER 108.6
GPER-sfGFP 112.35
XLHCGR 203.7
XLHCGR-sfGFP 144.7
STE12 114.05
Zs Green 122.05
G𝛂i 118.8

Table: Concentration of the DNA samples.

Results

The extracted DNA was stored in the freezer.

18th July 2019

1. 5th PCR amplification

Team members: Noël and Signe

After the PCRs we ran on the 5th, 8th and 9th showed unsatisfying results, we decided to repeat the PCR amplification of the human LH GPCR receptors, using a high fidelity buffer instead of X7 buffer.

Materials and procedure
  1. The same protocol as the previous days was followed (protocol from July 5th), only exchanging the buffer from 10X X7 PCR buffer with 5X HF buffer. Since this buffer is only half as concentrated than the other one, twice of it and a respectively adjusted amount of water has to be used.
  2. The following samples were PCR-amplified and loaded on the gel.
    1. Hu-LHCGR
    2. Hu-LHCGR-linker
    3. Negative control (no template)
Results

Gel loaded with the following aliquots from left to right: Marker, HuLHGPCR, huLHGPCR-linker, negative control, empty, Marker.

The results mirrored the earlier issues with many shorter unspecific bands. After a discussion with supervisor Karell, we should try temperature optimizing with the next PCR. If we still have issues, we can cut out bands from a gel. For this, we can use the PCR results from 9th of July, which seemingly had high amounts of amplified fragments of the desired size (2kbp).

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We are Ovulaid: a team of 13 students from the University of Copenhagen working on a novel ovulation detection system, using synthetic biology.

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