User:Elaine Marie Robbins/Notebook/CHEM-496/2011/11/02: Difference between revisions

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<b>DNA Transformation II</b>
<b>DNA Transformation II</b>


To transform DNA coding for mutant GFP (mutated on [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/20|09/20/11]]) into Novablue cells.
To transform DNA coding for mutant GFP (mutated on [[User:Elaine Marie Robbins/Notebook/CHEM-496/2011/11/01|11/01/11]]) into Novablue cells.




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<b>AuNP VIII</b>
<b>AuNP VIII</b>


# Combine 8 mL of water, 1 mL HAuCl<sub>4</sub> (2.9 mM), and 1 mL BSA (1.55 μM)in that order a test tube.
# Combine 8 mL of water, 1 mL HAuCl<sub>4</sub> (2.9 mM), and 1 mL BSA (1.55 μM) in that order 3 test tubes.
# Mix one test tube.
# Place test tubes in an oven at 80°C for 30 min.
# Place test tubes in an oven at 80°C for 30 min.
# Remove test tubes from oven for 10 min.
# Remove 2 test tubes from oven for 10 min, leave one in the oven.
# Repeat steps 2 and 3 for 3.5 hr.
# Repeat steps 3 and 4 for 3.5 hr.




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# Digest non-methylated DNA with 1 μL Dpnl.
# Digest non-methylated DNA with 1 μL Dpnl.
# Make LB/agar plates
# Make LB/agar plates:
## Mix 0.875 g LB, 0.7 g agar, and 35 mL of water.
## Mix 0.875 g LB, 0.7 g agar, and 35 mL of water.
## Autoclave.
## Autoclave.
## Add 35 μL ampicillin before agar completely cools.
## Add 35 μL ampicillin before agar completely cools.
## Pour agar onto plate and allow to solidify.
# Place sterile tube and DNA in ice bucket for 15 min.
# Place sterile tube and DNA in ice bucket for 15 min.
# Add 5 μL of DNA and 40 μL of cells to the bottom of the tube.
# Add 5 μL of DNA and 30 μL of cells to the bottom of the tube.
# Incubate on ice for 30 min.
# Incubate on ice for 30 min.
# Heat shock DNA/cells at 42°C for 30 s.
# Heat shock DNA/cells at 42°C for 30 s.
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<b>Gel II</b>
<b>Gel II</b>


==Description==
# Make a 1% agarose gel by adding 0.25 g of 0.01 g/mL of agarose to 25 mL of 1x tris base, acetic acid, and TAE buffer and heating the mixture in the microwave for 40 s.
 
# Make a 1% agarose gel by adding 0.25 g of 0.01 g/mL of agarose to 25 mL of 1x tris base, acetic acid, and ethylenediaminetetraacetic acid (TAE) buffer and heating the mixture in the microwave for 40 s.
# Mix 2 μL of 6x gel loading dye with 10 μL of the DNA sample.
# Mix 2 μL of 6x gel loading dye with 10 μL of the DNA sample.
# Prepare a ladder containing DNA, glycerol, and gel loading dye.
# Prepare a ladder containing DNA, glycerol, and gel loading dye.
# Load DNA solutions and run the gel at 80 V for 40 min.
# Load DNA solutions and run the gel at 80 V for 40 min.
# Remove gel and place in TAE and ethidium bromide (EtBr) solution, mix for 15 min.
# Remove gel and place in TAE and EtBr solution, mix for 15 min.
# Rinse gel with TAE for 5 min.
# Rinse gel with TAE for 5 min.


==Data==
==Data==


<b>AuNP VIII</b>


At 60 min, gray fibers began to conglomerate in the test tubes that were removed from the oven. At approximately the same time, gray fibers began to appear throughout the solution left in the oven.


==Notes==
==Notes==
<b>AuNP VIII</b>
New HAuCl<sub>4</sub> was used to make the nanoparticle solution.
<b>Gel II</b>


Order of DNA gel:
Order of DNA gel:

Revision as of 11:24, 2 November 2011

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Objective

AuNP VIII

To synthesize AuNP using the procedure developed here:


Bakshi, Mandeep S.; Kaur, Harpreet; Khullar, Poonam, Banipal, Tarlok, S.; Kaur, Gurinder; Singh, Narpinder Journal of Physical Chemistry C 2011, 115(7), 2982-2992

http://pubs.acs.org/doi/abs/10.1021/jp110296y


In addition, to repeat the experiment performed on 08/31/11, 09/07/11, 09/27/11, 09/28/11, 10/19/11, 10/26/11, and 11/01/11.


DNA Transformation II

To transform DNA coding for mutant GFP (mutated on 11/01/11) into Novablue cells.


In addition, to repeat the experiment performed on 09/27/11.


Gel II

To determine whether or not the PCR performed on 11/01/11 of the DNA coding for a mutant GFP was successful using agarose gel electrophoresis.


In addition, to repeat the experiment performed on 09/21/11.

Description

AuNP VIII

  1. Combine 8 mL of water, 1 mL HAuCl4 (2.9 mM), and 1 mL BSA (1.55 μM) in that order 3 test tubes.
  2. Mix one test tube.
  3. Place test tubes in an oven at 80°C for 30 min.
  4. Remove 2 test tubes from oven for 10 min, leave one in the oven.
  5. Repeat steps 3 and 4 for 3.5 hr.


DNA Transformation II

  1. Digest non-methylated DNA with 1 μL Dpnl.
  2. Make LB/agar plates:
    1. Mix 0.875 g LB, 0.7 g agar, and 35 mL of water.
    2. Autoclave.
    3. Add 35 μL ampicillin before agar completely cools.
    4. Pour agar onto plate and allow to solidify.
  3. Place sterile tube and DNA in ice bucket for 15 min.
  4. Add 5 μL of DNA and 30 μL of cells to the bottom of the tube.
  5. Incubate on ice for 30 min.
  6. Heat shock DNA/cells at 42°C for 30 s.
  7. Incubate on ice for 5 min.
  8. Add 250 μL of SOC media.
  9. Incubate in shaker at 37°C for 1 hr.
  10. Spread 100 μL of cells on LB/agar plate.
  11. Store plate (inverted) in 37°C oven overnight.


Gel II

  1. Make a 1% agarose gel by adding 0.25 g of 0.01 g/mL of agarose to 25 mL of 1x tris base, acetic acid, and TAE buffer and heating the mixture in the microwave for 40 s.
  2. Mix 2 μL of 6x gel loading dye with 10 μL of the DNA sample.
  3. Prepare a ladder containing DNA, glycerol, and gel loading dye.
  4. Load DNA solutions and run the gel at 80 V for 40 min.
  5. Remove gel and place in TAE and EtBr solution, mix for 15 min.
  6. Rinse gel with TAE for 5 min.

Data

AuNP VIII

At 60 min, gray fibers began to conglomerate in the test tubes that were removed from the oven. At approximately the same time, gray fibers began to appear throughout the solution left in the oven.

Notes

AuNP VIII

New HAuCl4 was used to make the nanoparticle solution.


Gel II

Order of DNA gel:

Ladder|Tamra 1|Tamra 2|Group 1|Group 2|Group 3|Group 4|Group 5



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