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

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(Procedure)
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.
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Revision as of 21:01, 23 October 2012

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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 water40.6
10X cloned Pfu buffer5
25 mM dNTPs0.4
100 ng/μL DNA template1
Forward primer (K110 f)1
Reverse primer (K110A r)1
2.5 U/μL PfuTurbo DNA polymerase1
  • 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.


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