E.coli cells that expressed the desired construct were harvested. The instructions of the EZNA purification kit were followed. The final concentrations after the elution were measured via Nanodrop.
Results
Sample
DNA concentration [ng/µl]
XI2A
237.7
XI2C
386.5
10th August 2019
1. Primers and g-blocks dilution
Team members: Noël and Hitesh
Dilution of different g-blocks and primers that are necessary for building the kill switch.
Materials
G-blocks: BAX and BI-1
Primers: U-BAX-F, U-BAX-R, U-BI-1-F and U-BI-1-R
Milli-Q-Water
Procedure
For primer dilution:
Took the samples delivered by the IDT in powder form to dilute in Milli-Q-water and make a final stock solution to 100 μM. To do that, the OD values were given in nM concentrations and amount of water to be added to make a final concentration of 100 μM was calculated as in Table 1.
Further took a part of the Stock solution to make a working solution of 10 μM. To do so, took 10 μL of each stock solution and added 90 μL of water to it.
For gene fragment dilution:
The gene fragments supplied were 500 ng each, we added 50 μl of Milli-Q-water to each sample (BAX and BI-1)) to make a final concentration of 10ng/μL.
Primer dilution:
Primers
OD value
Water added
1. U-BAX-F
22.9 nmol
353 μl
2. U-BAX-R
35.3 nmol
229 μl
3. U-BI-1-F
33.7 nmol
337 μl
4. U-BI-1-R
35 nmol
350 μl
2. PCR amplification of BAX and BI-1/h2>
Materials
10X X7 PCR buffer
dNTP
F-primers (10 μM)
R-primers (10 μM)
Template
X7-Polymerase
mQ Water
Samples: XLHCGR, GPER, GPA1-Gαi & GPA1-Gαs
pCCW12
Procedure
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.
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.
Then, we pipette out the individual quantities of Master-mix into each labelled PCR tube.
Now added the primers specific to each PCR sample and finally the template specific for each PCR sample.
Gently mix the final sample, vortex before setting up the PCR machine.
Also, prepare a negative control which includes the entire mix except for the template.
Put the samples into the machine and select the program based on the requirement finally start the program.
PCR program 30 cycles of:
98 °C: 30 seconds
98 °C: 20 seconds
55 °C: 25 seconds
72 °C: 2 minutes and 30 seconds
72 °C: 5 minutes
12 °Cs: Infinity
3. Gel electrophoresis
Materials
Samples
DNA ladder
Loading dye
Procedure
To prepare the gel:
1% agarose gel was taken from the common stock in electrophoresis room
The Agarose-TAE buffer solution was poured into the casting tray. Please label the cast with iGEM after adding the comb to the solution.
Setting up the electrophoresis chamber:
Solidified gel was placed on the electrophoresis chamber filled with TAE buffer
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.
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.
Finally the electrophoresis setup was put to 100 V in the electric chamber and was run for 25 mins.
Results
Only BAX got amplified and not BI-I
4. PCR clean up
Materials
PCR clean up kit
Samples
Procedure
PCR clean up was carried out as per the information given in the E.N.Z.A PCR clean up kit.
Results
Clean up was done and the concentration was measured using nanodrop. The concentrations are as follows: BAX - 38.5 ng/μl
iGEM Team Copenhagen
iGEM_Copenhagen
iGEM_Copenhagen
UCPH.IGEM2019@gmail.com
