User:Floriane Briere/Notebook/CHEM-496/2011/09/27: Difference between revisions
(9 intermediate revisions by the same user not shown) | |||
Line 27: | Line 27: | ||
# Add 1µL of DpnI enzyme to the PCR solution (which containe the wild-type DNA) | # Add 1µL of DpnI enzyme to the PCR solution (which containe the wild-type DNA) | ||
# Make the LB/agar plate: | # Make the LB/agar plate: | ||
## The 200ml stock solution has been made by adding Agar (5g) and LB powder (5g) | ## The 200ml stock solution has been made by adding Agar (5g) and LB powder (5g); the agar/LB plate is made with LB powder which is full of nutriments; it will allow cells to live and grow | ||
## Let the solution become solid | ## Let the solution become solid | ||
## Place the solution into the oven to make it become liquid again | ## Place the solution into the oven to make it become liquid again | ||
Line 37: | Line 37: | ||
## Add 5µL of DNA solution to 40µL of cells solution | ## Add 5µL of DNA solution to 40µL of cells solution | ||
## Incubate on ice during 30 minutes | ## Incubate on ice during 30 minutes | ||
## Place the solution at 42°C for 30 seconds to make a heat shock | ## Place the solution at 42°C for 30 seconds to make a heat shock ; the heat shock favors the transformation of the DNA into the cells | ||
## Incubate in ice for 5 minutes | ## Incubate in ice for 5 minutes | ||
## Add 250μL of SOC media | ## Add 250μL of SOC media | ||
Line 44: | Line 44: | ||
# Store plate (inverted) at 37°C in a oven during 24 hours | # Store plate (inverted) at 37°C in a oven during 24 hours | ||
== | ==Results: Gold NPs synthesis== | ||
* Curve of the Absorbance as a function of the wavelength: | |||
[[Image:27sept - Absorbance =f(wavelength).png]] | |||
* Curve of the Absorbance as a function of the wavelength (zoomed in the 550nm area): | |||
[[Image:27sept - Absorbance =f(wavelength) - zoom.png]] | |||
* Curve of the Absorbance at 550nm as a function of time: | |||
[[Image:27sept - Abs(550nm) = f(time).png]] | |||
* Conclusion: | |||
At the end of the experiment, we obtained an incolore solution. | |||
We can observe that there is no significant increase of the Absorbance at 550nm during the experiment. Compare to previous results, we can say that Gold NPs synthesis is not efficient in Tris buffer. This result is quite similar to the one we obtained when we used acetate as a buffer (on the 7th of september). | |||
We obtained better results when we used water as a buffer (on the 31st of august). | |||
The next experiment is to make the same protocol, using water as a buffer, but with a stable temperature. | |||
==Results: DNA's transfer into cells== | |||
The first step of the experiment was to add Dpnl enzyme to the PCR solution. This step allowed us to digest unmethylated DNA; we selected methylated DNA for the transfer. | |||
Then, we transferred the DNA into novablue cells; cells which integrate our DNA molecules are then selected by adding Ampicillin to the agar/LB plate. In fact, the DNA molecule contains an antibiotics resistant gene. | |||
The cells were then placed into a favorable environment to allow them to grow up. | |||
After 24hours of incubation, we observed no cells on our plate. | |||
[[Category:Course]] | [[Category:Course]] |
Revision as of 11:50, 19 October 2011
File:BDLlogo notext ir.png Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectiveWe are going to conduct two different experiments:
Protocol
Results: Gold NPs synthesis
At the end of the experiment, we obtained an incolore solution. We can observe that there is no significant increase of the Absorbance at 550nm during the experiment. Compare to previous results, we can say that Gold NPs synthesis is not efficient in Tris buffer. This result is quite similar to the one we obtained when we used acetate as a buffer (on the 7th of september). We obtained better results when we used water as a buffer (on the 31st of august). The next experiment is to make the same protocol, using water as a buffer, but with a stable temperature. Results: DNA's transfer into cellsThe first step of the experiment was to add Dpnl enzyme to the PCR solution. This step allowed us to digest unmethylated DNA; we selected methylated DNA for the transfer. Then, we transferred the DNA into novablue cells; cells which integrate our DNA molecules are then selected by adding Ampicillin to the agar/LB plate. In fact, the DNA molecule contains an antibiotics resistant gene. The cells were then placed into a favorable environment to allow them to grow up. After 24hours of incubation, we observed no cells on our plate.
|