User:Michael F. Nagle/Notebook/Chem 571/2012/10/16: Difference between revisions
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== | ==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 [[AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol|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 | |||
**[[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/10/16|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 95<sup>o</sup>C | |||
***Repeat the following 30 times: | |||
****95<sup>o</sup>C for 30 seconds | |||
****-51<sup>o</sup>C for 30 seconds | |||
****72<sup>o</sup>C for 60 seconds | |||
***Maintain at 72<sup>o</sup>C | |||
***Slowly cool to 0<sup>o</sup>C | |||
**Solutions were stored in the freezer. | |||
==HRP Chemiluniscence Assay== | |||
*[[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological_Chemistry_I/2012/10/16]] prepared 30mM luminol stock. Iodophenol, HRP and H<sub>2</sub>O<sub>2</sub> stock prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02|10/2]] were used. | |||
*The reaction solutions prepared contained .18mM iodophenol, .1mM H2O2 and 3mM luminol in 1mL quartz cuvettes. | |||
*2mM HRP added to the solution did not produce luminescence, nor did 10mM. | |||
*20mM produced a blue light that faded over the course of 3 minutes. | |||
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Revision as of 00:45, 7 December 2012
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Objectives
PCR Mutation
HRP Chemiluniscence Assay
|