Toronto/Lab Protocols/Quantitation
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== Quantitation == | == Quantitation == | ||
| - | #Take the extracted DNA and run a gel to find out the concentration (per μL) of your DNA. | + | # 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 plasmid + 4 μL ddH2O + 1 μL Loading Dye |
| - | + | #* 1 μL insert + 4 μL ddH2O + 1 μL Loading Dye | |
| - | + | {| style="text-align:center;" align="center" | |
| - | + | ! Gel Lane !! Fragment | |
| - | + | |- | |
| - | + | | 1 || HindIII Ladder (5 μL) | |
| - | + | |- | |
| - | + | | 2 || HindIII Ladder (2 μL) | |
| - | + | |- | |
| - | + | | 3 || HindIII Ladder (1 μL) | |
| - | + | |- | |
| - | #Ensure that the plasmid and inserts are at the correct lengths using the 1 Kbp ladder. | + | | 4 || Blank |
| - | #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. | + | |- |
| + | | 5 || Plasmid (All of it) | ||
| + | |- | ||
| + | | 6 || Blank | ||
| + | |- | ||
| + | | 7 || Insert (All of it) | ||
| + | |- | ||
| + | | 8 || Blank | ||
| + | |- | ||
| + | | 9 || 1 Kbp Ladder (3 μL) | ||
| + | |} | ||
| + | # | ||
| + | # 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] | ||
Revision as of 03:48, 27 October 2007
Quantitation
- 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) |
- 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 BlueGenes
