Toronto/Lab Protocols/Quantitation

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== Quantitation ==
== Quantitation ==
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#Take the extracted DNA and run a gel to find out the concentration (per μL) of your DNA.
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# Take the extracted DNA and run a gel to find out the concentration (per μL) of your DNA.
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*1 μL plasmid + 4 μL ddH2O + 1 μL Loading Dye
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#* 1 μL plasmid + 4 μL ddH<sub>2</sub>O + 1 μL Loading Dye
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          * *      Gel Lane Fragment
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#* 1 μL insert + 4 μL ddH<sub>2</sub>O + 1 μL Loading Dye
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      1 HindIII Ladder (5 μL)
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{| style="text-align:center;" border=1 align=center
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      2 HindIII Ladder (2 μL)
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! Gel Lane !! Fragment
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      3 HindIII Ladder (1 μL)
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|-
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      4 Blank
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|      1 || HindIII Ladder (5 μL)
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      5 Plasmid (All of it)
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|-
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      6 Blank
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|      2 || HindIII Ladder (2 μL)
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      7 Insert (All of it)
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|-
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      8 Blank
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|      3 || HindIII Ladder (1 μL)
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      9 1 Kbp Ladder (3 μL)
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|-
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#Ensure that the plasmid and inserts are at the correct lengths using the 1 Kbp ladder.
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|      4 || Blank
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#Note that 2 μL of HindIII will twice as bright as 1 μL of HindIII, and 5 μL of HindIII will be five times as bright. So to determine concentration, estimate the intensity of the bands compared with the 3 HindIII ladders and assign it a relative intensity factor. Read off the concentration value on the Ladder card, and multiply by the relative intensity factor to determine your sample concentration in ng/μL.
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|-
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|      5 || Plasmid (All of it)
 +
|-
 +
|      6 || Blank
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|-
 +
|      7 || Insert (All of it)
 +
|-
 +
|      8 || Blank
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|-
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|      9 || 1 Kbp Ladder (3 μL)
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|}
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#
 +
# Ensure that the plasmid and inserts are at the correct lengths using the 1 Kbp ladder.
 +
# Note that 2 μL of HindIII will twice as bright as 1 μL of HindIII, and 5 μL of HindIII will be five times as bright. So to determine concentration, estimate the intensity of the bands compared with the 3 HindIII ladders and assign it a relative intensity factor. Read off the concentration value on the Ladder card, and multiply by the relative intensity factor to determine your sample concentration in ng/μL.
Jump to [http://igem.skule.ca/lab/protocols/quantitation.htm BlueGenes]
Jump to [http://igem.skule.ca/lab/protocols/quantitation.htm BlueGenes]

Latest revision as of 03:57, 27 October 2007

Quantitation

  1. Take the extracted DNA and run a gel to find out the concentration (per μL) of your DNA.
    • 1 μL plasmid + 4 μL ddH2O + 1 μL Loading Dye
    • 1 μL insert + 4 μL ddH2O + 1 μL Loading Dye
Gel Lane Fragment
1 HindIII Ladder (5 μL)
2 HindIII Ladder (2 μL)
3 HindIII Ladder (1 μL)
4 Blank
5 Plasmid (All of it)
6 Blank
7 Insert (All of it)
8 Blank
9 1 Kbp Ladder (3 μL)
  2. Ensure that the plasmid and inserts are at the correct lengths using the 1 Kbp ladder.
  3. Note that 2 μL of HindIII will twice as bright as 1 μL of HindIII, and 5 μL of HindIII will be five times as bright. So to determine concentration, estimate the intensity of the bands compared with the 3 HindIII ladders and assign it a relative intensity factor. Read off the concentration value on the Ladder card, and multiply by the relative intensity factor to determine your sample concentration in ng/μL.

Jump to [http://igem.skule.ca/lab/protocols/quantitation.htm BlueGenes]

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