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

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 13: Line 13:
* Run HRP Luminol Assay
* Run HRP Luminol Assay


==Procedure==
==Preparation==
* Assigned Primer= ADA K110A F (forward primer)  
* Assigned Primer= ADA K110A F (forward primer)  
** Sequence Issue number: 87635082
** Sequence Issue number: 87635082
Line 21: Line 21:
**33.4 nm= .56 mg ( amount of present plasmid)  
**33.4 nm= .56 mg ( amount of present plasmid)  


concentration of plasmid needed for experiment= 100ng/μL
Concentration of plasmid needed for experiment= 100ng/μL
 
* Calculations
.56mg= 5600 ng
.56mg= 5600 ng
M<sub>1</sub>V<sub>1</sub>= M<sub>2</sub>V<sub>2</sub>
M<sub>1</sub>V<sub>1</sub>= M<sub>2</sub>V<sub>2</sub>
5600 ng/μL x V<sub>1</sub>= 100ng x 1000μL   
5600 ng/μL x V<sub>1</sub>= 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.  
*(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.  
V<sub>2</sub>= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL
V<sub>2</sub>= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL
==Procedure==
# 1mL of deionized water was added to .56mg of ADA K110A F
# 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.
# Next, 2 50μL solutions were made with the following components in microcentrifuge tubes. The DNA polymerase was added last.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''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.
# Heat for 2 minutes at 95°C.
# Heat for 30 seconds at 95°C.
# Heat for 30 seconds at 57°C.
#  Heat for 8 min at 72°C.
#  Repeat steps two through four 30 times.
#  Heat for 10 minutes at 72°C.
#  Chill at 0°C until the samples needed again.


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 18:01, 23 October 2012

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/10/16&oldid=640241"