Team:UCopenhagen/Notebook/Week 27







Week 27 (1st-7th of July)

1st of July

1. Normal PCR amplification sample 1,2, 11 and 12

Team members:

To start the USER ligation all over for samples 1 (GPER), 2 (XLHCGR) , 11 (GPER-Linker) and 12 (XLHGCR-Linker), we amplified the original fragments from IDT by PCR. To amplify the gene sequences for sample 1, 2, 11 and 12 (number as labelled on 22nd May 2019). For samples 11 and 12, the GPER and XLHCGR fragments were used, respectively. The Linkers are attached to the reverse primers.

Materials
Materials Quantity (μL)
10X X7 PCR buffer 5
dNTPs 4
F-primer (10 μM) 2.5
R-primer (10 μM) 2.5
Template 0.5
X7-Polymerase (Add last) 0.5
mQ Water 35
Total 50
Procedure
  1. Noted the number of samples to be PCR amplified and calculated the quantities of each material to be added for the master-mix. It should be noted that the amount of water added is to make the total solution of 50 μL.
  2. The mastermix for PCR included the buffer, dNTPs, and water. Mixed the total quantity required based on the number of PCR amplification samples.
  3. Then, we pipette out the individual quantities of Master-mix into each labelled PCR tube.
  4. Now added the primers specific to each PCR sample and the template specific for each PCR sample. Finally we added the X7 polymerase in the end and maintained the sample cold.
  5. Gently mix the final sample, vortex before setting up the PCR machine.
  6. Also, prepare a negative control which includes the entire mix except for the template.
  7. Put the samples into the machine and select the program based on the requirement finally start the program.
  8. PCR program 30 cycles of:
    1. 98 degree celsius: 30 seconds
    2. 98 degree celsius: 20 seconds
    3. 55 degree celsius: 25 seconds
    4. 72 degree celsius: 2 minutes and 30 seconds
    5. 72 degree celsius: 5 minutes
    6. 12 degree celsius: Infinity
Data
SERIAL NO. FRAGMENT NUCLEOTIDE FRAGMENT F-PRIMER R-PRIMER
1 GPER GPER GPER-F GPER-R
2 XLHCGR XLHCGR XLHCGR-F XLHCGR-R
11 GPER-linker GPER GPER-F GPER-linker-R
12 XLHCGR-linker XLHCGR XLHCGR-F XLHCGR-linker-R
Results

In the experimental setup, the amplified sample numbers were labelled such and were arranged serially for further gel electrophoresis.

2. Gel electrophoresis for sample 1,2,11 and 12

Team members: Signe and Noel

To check for the amplified gene sequences the samples are loaded on a 1 % agarose gel were then run in gel electrophoresis and analysed under the “Gel-Doc”to confirm for positive amplification.

Materials
  • Agarose
  • 1X TAE-buffer
  • Loading dye
  • DNA ladder
  • PCR tubes
  • Ice box
Procedure
  1. To prepare the gel
    1. 1% agarose gel was taken from the common stock in electrophoresis room
    2. The Agarose-TAE buffer solution was poured into the casting tray. Please label the cast with iGEM after adding the comb to the solution.
  2. Setting up the electrophoresis chamber
    1. Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
    2. To prepare the sample to be loaded, mixed 4 μL of each sample was taken into a new PCR tube and 1 μL of loading dye was added to each sample.
    3. To load the wells, the first and the last well were filled with the 1 kb DNA ladder while wells 2 to 13 were loaded with the PCR samples (1-13) while 14 was loaded with negative control.
  3. Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.
Data
SERIAL NO. FRAGMENT
1 GPER
2 XLHCGR
11 GPER-linker
12 XLHCGR-linker
1 2 3 4 5 6 7
Ladder Sample 1 Sample 2 Sample 11 Sample 12 Negative control Ladder
Results

We can see that we have bands in well 2, 3 and 5 but not in 4 which means the PCR did not work for sample 11.

3. Plasmid DNA extraction of samples from Nat

Team members: Signe and Noel

We extracted plasmid DNA from liquid cultures Nat gave us, the samples are labelled such as mentioned in the table below. The E. coli colonies transformed with these respective vectors were in the plates labelled 1. X3A-pCCW12-GPER 2. X3A-pCCW12-GPER-sfGFP 3. X3A-pCCW12-XLHCGR-sfGFP 4. X3A-pCCW12-XLHCGR and the positively transformed colonies were grown in liquid LB medium overnight and labelled on the basis of the plate and the colony number as mentioned in the table with plate number following with a decimal and then the colony number.

Materials
  • E.Z.N.A. Plasmid DNA Mini Kit I.
  • Liquid culture of following 4 samples
    Name of the Insert Our name of the samples (corresponds to number of colony on the plate the liquid culture was inoculated with)
    X3A-pCCW12-GPER 1.1 and 1.2
    X3A-pCCW12-GPER-sfGFP 2.2 and 2.3
    X3A-pCCW12-XLHCGR-sfGFP 3.1 and 3.2
    X3A-pCCW12-XLHCGR 4.1 and 4.2
Procedure
  1. We followed the Plasmid purification as described in the instructions of the kit (see also “30th May 2019 16. Plasmid DNA Extraction”). We used 1 ml of each liquid culture and did not do any of the optional steps.
Results

The purified samples were stored in the freezer at -20 degree celsius.

4. E.coli incubation for vector backbone amplification

In the lab we have E. coli transformed with empty vector backbones, we took the stock solution of E. coli and incubated them in liquid culture for the amplification of vector backbones.

Materials
  • E. coli cells with plasmid backbones for X3A, Ass2A, Ass2B, Ass2C, BAss2, X3c
  • Liquid LB Agar medium
  • Carbenicillin
  • Falcon tube
Procedure
  1. Liquid LB agar medium 80 mL bottle was collected from stockroom.
  2. Added 160 uL of Carbenicillin into the entire Liquid LB agar bottle, labelled with date and selection marker.
  3. Poured 10mL of the medium into each falcon tube, one tube per E. coli. Sample with each vector backbone.
  4. Inoculated E. coli culture from plate into falcon tube
  5. Labelled and kept the falcon tubes into shaking incubator at 30 degree celsius
Results

The falcon tubes will further be taken for plasmid purification.

2nd of July

1. Preparing samples for sequencing

Team members: Signe and Noel

The plasmids purified for the samples 1. X3A-pCCW12-GPER 2. X3A-pCCW12-GPER-sfGFP 3. X3A-pCCW12-XLHCGR-sfGFP 4. X3A-pCCW12-XLHCGR that we extracted on 1st July 2019 were prepared for sequencing.

Materials
  • Samples that were prepared July 1st 2019
  • Primers
  • Waters
Procedure
  1. The concentration of each purified plasmid DNA sample was measured using NanoDrop.
  2. A stock solution of the primers was prepared by adding 10 µl of each primer to a new eppendorf tube containing 90 µl mH20, respectively. The stock solutions were stored in the freezer after usage.
  3. To assure that the whole constructs would be synthesised three primers were used for each vector (USER primer of the first gene and two further primers according to list in R148) Note: We used Sanger sequencing which is limited to the sequencing of approximately 700 bp at a time. Therefore an extra primer was used.
  4. For each construct only one sample was prepared for sequencing (Samples 1.1, 1.2, 2.2, 2.3, 3.1, 3.2, 4.1 and 4.2). Moreover the overview of samples prepared for sequencing is mentioned in the table The samples were prepared as follows: DNA: µl corresponding to 500 ng DNA, Primer: 3,3 µl, mH20: x µl
    5. , Reaching a total of 10 µl
  5. Stickers with QR codes. The stickers consists of two parts, the smaller of which was attached into the lab book in a list next to the corresponding construct and primer. The second part was wrapped around the eppendorf tubes in such a way that the QR code was clearly visible.
Data
The E coli colonies transformed with these respective vectors were in the plates labelled 1. X3A-pCCW12-GPER 2. X3A-pCCW12-GPER-sfGFP 3. X3A-pCCW12-XLHCGR-sfGFP 4. X3A-pCCW12-XLHCGR and the positively transformed colonies were grown in liquid LB medium overnight and labelled on the basis of the plate and the colony number as mentioned in the table with plate number following with a decimal and then the colony number.
Name of the Insert Our name of the samples (corresponds to number of colony on the plate the liquid culture was inoculated with)
X3A-pCCW12-GPER 1.1 and 1.2
X3A-pCCW12-GPER-sfGFP 2.2 and 2.3
X3A-pCCW12-XLHCGR-sfGFP 3.1 and 3.2
X3A-pCCW12-XLHCGR 4.1 and 4.2
Table shows nanodrop concentration for each sample and the calculated volume of template that needs to be added for sequencing in each sample.
Construct Sample Nr. A60/280 A260/230 ng/µl Avg ng/uL µl used
X3A-pCCW12-GPER 1.1 1.81 1.84 1.76 2.02 172.1 156.8 164.45 3.04
1.2 1.83 1.81 2.06 1.86 168.3 182.0 175.15 2.85
X3A-pCCW12-GPER-sfGFP 2.2 1.80 1.86 1.72 2.09 224.9 214.4 219.65 2.28
2.3 1.83 1.85 1.79 2.08 241.3 232.0 236.65 2.11
X3A-pCCW12-XLHCGR-sfGFP 3.1 1.84 1.87 2.28 2.24 207.3 202.2 204.7 2.44
3.2 1.83 1.86 2.17 2.22 211.1 203.5 207.3 2.41
X3A-pCCW12-XLHCGR 4.1 1.82 1.85 1.95 2.22 180.0 168.5 174.25 2.87
4.2 1.79 1.79 1.79 1.79 203.7 203.5 203.6 2.46
Overview of the sample preparation
Sample name (see “36 dated 1st July 2019”) Used Primers Volume per Primer solution in µL
1.1 and 1.2 Y81, Y74, GPER-F 1.1
2.2 and 2.3 Y81, Y74, GPER-F, GPER-R 0.825
3.1 and 3.2 Y81, Y74, XLH-F, XLH-R 0.825
4.1 and 4.2 Y81, Y74, XLH-F 1.1

Results

We made a mistake in the sequencing. Instead of making multiple samples with each primer we added all the primers in the same eppendorf tube and therefore got no data for the sequencing.

3rd of July

1. Competent cell test

Team members: Hitesh and Mads

To test the efficiency of the competent cells (E.coli) prepared

Materials
  • Competent cell test kit
  • Agar plates with Chloramphenicol (was not added last time)
Procedure
  1. The kit includes two vials of dried-down purified plasmid DNA from BBa_J04450 (RFP construct) in plasmid backbone pSB1C3. The plasmid backbone contains the gene for chloramphenicol resistance.
  2. When resuspended with 50µL dH2O, the vials will result in different concentrations: 100 pg/µL, 10 pg/µL. Perform transformations with each of these to determine how efficient your competent cells are.
  3. Standard E.coli transformation protocol can be used to test the competent cells.
  4. The transformed E.coli cells(10 pg/µL and 100 pg/µL) were plated and incubated overnight.
Results
The efficiency was calculated on 4th July 2019. The desired result is within 1.5 * 108 and 6 * 10.
Plasmid concentration No. of colonies Efficiency= (cfu/DNA amount in ng) * 1000 uL
100 pg/uL 21 42
10 pg/uL 2 0

2. Plasmid purification

Team members: Mads and Hitesh

To extract the DNA for samples X3A, BAss2, X3C, ASS2A, ASS2B and ASS2C from E.coli

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.
Data
Concentration of the DNA samples
Sample Concentration
X3A 403.3 ng/μl
B 287.5 ng/μl
X3C 286.8 ng/μl
ASS2A 198.4 ng/μl
ASS2B 164.8 ng/μl
ASS2C 219.7 ng/μl
Results

The extracted DNA was stored in the freezer.

4th of July

1. Gel electrophoresis

Team members: Jonas

Verify purified empty plasmid samples from 3rd July 2019

Materials
  • Extracted empty plasmid backbones from the 3rd of july
  • Gelred
  • 1 % agarose solution
Procedure
  1. The first step is to cast an agarose gel using a 1 % agarose solution
  2. 2 µl of the extracted DNA from yesterday was mixed with 1 µl of gelred
  3. After mixing the samples were run on the gel
Data
Well nr. Sample
1 ladder
2 Ass2c
3 Ass2b
4 Ass2a
5 x3c
6 x3b
7 x3a
8 H2O
9 nothing
10 ladder
Results

5th of July

1. 1st Human LHCGR PCR amplification: X7-PFU Polymerase and gel electrophoresis

Team members: Mads & Iben

We wanted to also use human LH receptor for our project, and To amplify the human LH receptor gene, amplification is performed with the reverse primer with or without a linker to make LH receptor gene. We also performed the dilution of primer sets for the human LHCGR as mentioned in the table below.

Materials
  • 10X X7 PCR buffer
  • dNTP
  • F-primers (10 μM): HuLHCGR-F
  • R-primers (10 μM): HuLHCGR-R and HuLHCGR-linker-R
  • Template
  • X7-Polymerase
  • mQ Water
  • Samples: HuLHCGR template
Procedure
  1. Noted the number of samples to be PCR amplified and calculated the quantities of each material to be added for the master-mix. It should be noted that the amount of water added is to make the total solution of 50 μL.
  2. The mastermix for PCR included the buffer, dNTPs, water and X7 polymerase. Mixed the total quantity required based on the number of PCR amplification samples.
  3. Then, we pipette out the individual quantities of Master-mix into each labelled PCR tube.
  4. Now added the primers specific to each PCR sample and finally the template specific for each PCR sample.
  5. Gently mix the final sample, vortex before setting up the PCR machine.
  6. Also, prepare a negative control which includes the entire mix except for the template.
  7. PCR program 30 cycles of:
    1. 98 degree celsius: 30 seconds
    2. 98 degree celsius: 20 seconds
    3. 55 degree celsius: 25 seconds
    4. 72 degree celsius: 2 minutes and 30 seconds
    5. 72 degree celsius: 5 minutes
    6. 12 degree celsius: Infinity
Data
Primer names Concentration IDT (nmol) Stock solution (10mM) Working stock (100mM)
HuLHCGR-F 31.5 315 uL of water 10 uL of stock with 90 uL of mWater
HuLHCGR-R 33.9 339 uL of water 10 uL of stock with 90 uL of mWater
HuLHCGR-linker-R 30.9 309 uL of water 10 uL of stock with 90 uL of mWater
The composition of materials to be used per PCR amplification sample
Materials Quantity (μL)
10X X7 PCR buffer 5
dNTPs 4
F-primer (10 μM) 2.5
R-primer (10 μM) 2.5
Template 0.5
X7-Polymerase 0.5
mQ Water ?
Total 50

1. Gel electrophoresis

Two gels was run on amplified PCR products from the morning of July 5th. The second was run, because of issues with the first as described in results.

Data
Wells from left: 1 huLH, 2 huLH, 3 huLH-linker, 4 huLH-linker, 5 NC
Wells from left: 1 NC, 2 huLH, 3 huLH-linker
Results

The gel showed several undesired bands and some of them were more clear than the desired. Furthermore, it seems that we have swapped the NC and one of the samples, since the amplified product is seen in the negative control in both gels. We do not know which sample we have swapped, and we can’t therefore continue with these amplified products, and will redo the samples.

About Us

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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iGEM Team Copenhagen

iGEM_Copenhagen

iGEM_Copenhagen

UCPH.IGEM2019@gmail.com

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