User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/16

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==Procedure==
==Procedure==
-
 
+
* Concentration needed to dilute the primer E34K reverse was calculated with the below information:
-
 
+
    Melting Temperature in 50mM NaCl=62.2°C
 +
    Molecular Weight=16,877.0g/mol
 +
    Amount of miligrams present=0.58mg
 +
    Strand: 5'-ATG TTA AATCTA AAT GGC AAT GTA ATT TAA CTT TGG GAA TTC TTC TTC ATA TTT C-3'
 +
    GC content=27.2%
 +
    nmoles/OD260=1.9
 +
    ug/OD260=31.6
 +
    Ext. Coefficient: 534,6000 L/(mole·cm)
 +
    Amount of moles=34.4moles
 +
* The concentration of primer E34K reverse was calculated, and amount of diluted primer needed for PCR was determined the following way:
 +
    Target concentration: 100ng/uL
 +
    0.58mg=0.58·10<sup>6</sup>ng
 +
    0.58·10<sup>6</sup>ng / 1000μL H<sub>2</sub>O = 580ng/μL
 +
    100ng/uL x 1000uL / 580ng/uL = 172.41μL
 +
**1mL of sterile water was added into reverse primer, making the concentration of the reverse primer 3.4×10<sup>4</sup>M.
 +
**172.41μL of 3.4×10<sup>4</sup>M reverse primer was pipetted into a 1.5mL microcentrifuge tube, and 827.58μL of sterile water was added to make up a total of 1mL 100ng/μL reverse primer.
 +
**100ng/μL reverse primer was made at 1mL.
 +
* With 100ng/μL of forward primer of E34K made, PCR solution was made with the following reactants:
 +
  1μL of forward E34K primer
 +
  1μL of reverse E34K primer
 +
  1μL of ADA wild strand
 +
  0.4μL of 25mM dNTPs
 +
  5μL of 10x cloned Pfu buffer
 +
  40.6μL of sterile water
 +
  1μL of 2.5μL Turbo DNA polymerase
 +
**Turbo DNA polymerase was added last and right before PCR solution was placed on Thermocycler
 +
**Two of PCR solutions were made
 +
*The Thermocycler was programed in the following way to detach and reanneal DNA to allow DNA amplification:
 +
  Heating for 2minutes at 95°C
 +
  30 times of the following procedure:
 +
      Heating for 30 seconds at 95°C
 +
      Cooling for 30 seconds at -5°C
 +
      Reheating back for 1 minute at 72°C
 +
  Maintaining temperature at 72°C
 +
  Cooling back to 0°C for plasma stability
 +
* New sets of Au/BSA solutions was made with the following ratios:
 +
  60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, 170
 +
**Stock solution of BSA at 3μM and HAuCl4 at 26mM was made the following way:
 +
  Molecular weight of BSA: 66,000g/mol
 +
  0.0398g of BSA in powder form was weighted out and diluted in 20mL of water
 +
  0.0398g BSA ÷ 66,000g/mol = 3.01×10<sup>-5</sup>M = 30.1μM BSA
 +
**Stock for HAuCl4 from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03|10/03/12]] was used due to long shelf life
 +
* The amount of HAuCl4, BSA, and water was calculated via excel to make Au/BSA solutions with above ratios, show in table below:
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Revision as of 17:54, 26 October 2012

Experimental Biological Chemistry I Main project page
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Purpose

  • Perform polymerase chain reaction to design ADA plasmids with mutation at the 34th amino acid position from glutamate to lysine: E34K.
  • Make new Au/BSA solutions for more fibers in order to perform Atomic Absorption spectroscopy to find out gold concentration in solution

Procedure

  • Concentration needed to dilute the primer E34K reverse was calculated with the below information:
   Melting Temperature in 50mM NaCl=62.2°C
   Molecular Weight=16,877.0g/mol
   Amount of miligrams present=0.58mg
   Strand: 5'-ATG TTA AATCTA AAT GGC AAT GTA ATT TAA CTT TGG GAA TTC TTC TTC ATA TTT C-3'
   GC content=27.2%
   nmoles/OD260=1.9
   ug/OD260=31.6
   Ext. Coefficient: 534,6000 L/(mole·cm)
   Amount of moles=34.4moles
  • The concentration of primer E34K reverse was calculated, and amount of diluted primer needed for PCR was determined the following way:
   Target concentration: 100ng/uL
   0.58mg=0.58·106ng
   0.58·106ng / 1000μL H2O = 580ng/μL
   100ng/uL x 1000uL / 580ng/uL = 172.41μL
    • 1mL of sterile water was added into reverse primer, making the concentration of the reverse primer 3.4×104M.
    • 172.41μL of 3.4×104M reverse primer was pipetted into a 1.5mL microcentrifuge tube, and 827.58μL of sterile water was added to make up a total of 1mL 100ng/μL reverse primer.
    • 100ng/μL reverse primer was made at 1mL.
  • With 100ng/μL of forward primer of E34K made, PCR solution was made with the following reactants:
  1μL of forward E34K primer
  1μL of reverse E34K primer
  1μL of ADA wild strand
  0.4μL of 25mM dNTPs
  5μL of 10x cloned Pfu buffer
  40.6μL of sterile water
  1μL of 2.5μL Turbo DNA polymerase
    • Turbo DNA polymerase was added last and right before PCR solution was placed on Thermocycler
    • Two of PCR solutions were made
  • The Thermocycler was programed in the following way to detach and reanneal DNA to allow DNA amplification:
  Heating for 2minutes at 95°C
  30 times of the following procedure:
     Heating for 30 seconds at 95°C 
     Cooling for 30 seconds at -5°C 
     Reheating back for 1 minute at 72°C 
  Maintaining temperature at 72°C 
  Cooling back to 0°C for plasma stability
  • New sets of Au/BSA solutions was made with the following ratios:
  60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, 170
    • Stock solution of BSA at 3μM and HAuCl4 at 26mM was made the following way:
  Molecular weight of BSA: 66,000g/mol
  0.0398g of BSA in powder form was weighted out and diluted in 20mL of water
  0.0398g BSA ÷ 66,000g/mol = 3.01×10-5M = 30.1μM BSA
    • Stock for HAuCl4 from 10/03/12 was used due to long shelf life
  • The amount of HAuCl4, BSA, and water was calculated via excel to make Au/BSA solutions with above ratios, show in table below:


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