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

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AuNP VIII, DNA Transformation II, Gel II <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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.
  3. Place sterile tube and DNA in ice bucket for 15 min.
  4. Add 5 μL of DNA and 40 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

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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