User:Michael F. Nagle/Notebook/Chem 571/2012/10/16

From OpenWetWare

< User:Michael F. Nagle | Notebook | Chem 571 | 2012 | 10(Difference between revisions)
Jump to: navigation, search
(HRP Chemiluniscence Assay)
Current revision (03:45, 7 December 2012) (view source)
 
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 +
==Objectives==
 +
*Mutate ADA via PCR mutation
 +
**Mutated ADA is intended for nucleation of AuNPs
 +
*Produce chemiluminescence by adding luminol to HRP assay
==PCR Mutation==
==PCR Mutation==
* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|PCR Mutation]] was followed  
* The procedure for [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|PCR Mutation]] was followed  

Current revision

Project name Main project page
Previous entry      Next entry

Objectives

  • Mutate ADA via PCR mutation
    • Mutated ADA is intended for nucleation of AuNPs
  • Produce chemiluminescence by adding luminol to HRP assay

PCR Mutation

  • The procedure for PCR Mutation was followed
    • The specifications of the forward primer used are as follows:

Image:Primer212.png

    • 1mL molecular biology grade water was added to the primer.
    • The volume of primer solution needed to prepare 1mL of 100ng/uL solution was calculated
      • 0.49mg/1000μL = 490ng/μL
      • 100ng/uL*1000uL / 490ng/uL = 204.08μL
    • Keyun Wang prepared 100ng/uL solution of the matching forward primer.
    • Two solutiosn were prepared with the following:
      • 1uL forward primer
      • 1uL reverse primer
      • 1uL wild strand ADA
      • 5uL 10x Pfu buffer
      • 40.6uL molecular biology grade water
      • 0.4Turbo DNA polymerase 25mM dNTPs
      • 1uL Turbo DNA polymerase solution added immediately before placing on thermocycler
    • Thermocycler was programmed as following:
      • Heat for 2 minutes at 95oC
      • Repeat the following 30 times:
        • 95oC for 30 seconds
        • -51oC for 30 seconds
        • 72oC for 60 seconds
      • Maintain at 72oC
      • Slowly cool to 0oC
    • Solutions were stored in the freezer.

HRP Chemiluniscence Assay


Personal tools