User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/16

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PCR of ADA Mutations

Goals

  • Prepare ADA primers in order to conduct a polymerase chain reaction (PCR)
  • Run HRP Luminol Assay

Preparation

  • Assigned Primer= ADA K110A F (forward primer)
    • Sequence Issue number: 87635082
    • Sequence: ATT TGG CCA AAT GGG CAG TTA TTG AA
    • TM= 65.5°C
    • MW= 16833.0 g/mol
    • 33.4 nm= .56 mg ( amount of present plasmid)

Concentration of plasmid needed for experiment= 100ng/μL

  • Calculations

.56mg= 5600 ng M1V1= M2V2 5600 ng/μL x V1= 100ng x 1000μL

  • (The container containing the plasmid only had room for 1mL of water to be added- as a result, a dilution needed to be done in order to get the appropriate concentration.

V2= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL

Procedure

  1. 1mL of deionized water was added to .56mg of ADA K110A F
  2. 17.86μL of this solution was pipetted out and added to a centrifuge tube. 982.14μL of autoclaved water was then added to this sample in order to dilute it to 100ng/μL.
  3. Next, 2 50μL solutions were made with the following components in microcentrifuge tubes. The DNA polymerase was added last.
Component Volume [μL]
Distilled water 40.6
10X cloned Pfu buffer 5
25 mM dNTPs 0.4
100 ng/μL DNA template 1
Forward primer (K110 f) 1
Reverse primer (K110A r) 1
2.5 U/μL PfuTurbo DNA polymerase 1
  • Immediately after the addition of the DNA polymerase, the two sample solutions were placed in a thermocycler. The heating and cooling cycle of the thermocycler for the samples is as follows.
  1. Heat for 2 minutes at 95°C.
  2. Heat for 30 seconds at 95°C.
  3. Heat for 30 seconds at 57°C.
  4. Heat for 8 min at 72°C.
  5. Repeat steps two through four 30 times.
  6. Heat for 10 minutes at 72°C.
  7. Chill at 0°C until the samples needed again.
  • a new stock of luminol solution was made at a pH of 10.56. For full details on preparation, calculation, and concentration please see Mary's Notebook
  • The other reagents were either left over from the HRP absorbance assay from another week or were remade in using the same method.
Reagent Desired concentration in cuvet M1 V1 M2 V2 ' HRP '
Iodophenol .18mM 10mM 27μL .18mM 1500μL Concentration in Cuvet Volume
H202 .1mM 1.7mM 88μL .1mM 1500μL 20mM 30μL
Luminol 3mM 30mM 450μL 3mM 1500μL 22mM 33μL
24mM 36μL
26mM 39μL
28mM 42μL

Calculations

  • Concentrations of the stock solutions:
    • 10mM iodophenol in Dimethyl Sulfoxide (DMSO)
    • 1.7mM H202
    • 30mM luminol
    • 1mg/mL HRP

Total Volume in cuvet= 1500μL

Conclusions

  • The goal for today was the run the HRP chemiluminescence assay. Last week there had been no emittance from the sample. The luminol pH had been at 8 and after researching the literature, we decided that it was necessary to increase the pH of luminol in order to push forward a reaction. As a result, we aimed to make a luminol solution with a pH of 10.5. The actual pH of the luminol was 10.56.
  • We also decided that we needed to increase the concentration of the HRP in the cuvet. The Fluorimeter was not working and so we just used visual analysis to see if any fluorescence was occurring. The first concentration in the cuvet we tested was 2mM- which did not produce any luminescence. The next concentration we attempted was 10mM which also did not result in any luminescence. We then decided to double this final concentration to 20mM HRP while maintaining 3mM concentration of Luminol. This sample resulted in fluorescence of the luminol in the form of a blue light! We plan on running different variations of the HRP concentration beginning with 20mM tomorrow when conducting the chemiluminescence assay. All other reagents will remain constant.