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Team:TU-Delft/Notebooks/Protocols/PCR
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=='''Protocols''' == | =='''Protocols''' == | ||
| - | ==='''PCR'''=== | + | ===''' Colony PCR'''=== |
| - | + | 1. Make biobrick mastermix, containing per sample | |
| + | * 12.5 ul Taq mastermix | ||
| + | * 2.5 ul 10x forward biobrick primer | ||
| + | * 2.5 ul 10x reverse biobrick primer | ||
| + | * 7.5 ul H2O | ||
| - | + | 2. Put 25 ul in the PCR tubes. | |
| - | + | ||
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| - | + | ||
| - | + | ||
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| - | PCR using Taq Mastermix | + | 3. With a toothpick or pipet point, touch a colony and stir it through the fluid |
| + | |||
| + | 4. Run the iGEM colpcr program (to be added later) | ||
| + | |||
| + | ==='''PCR using Taq Mastermix'''=== | ||
Contents of the PCR mix is the for a large part the same as mentioned above for the Colony PCR. Differences will be noted here. First, instead of biobrick primer, any primer of choice can be added, also 2.5ul if standard solution has a concentration of 10 pmol/ul. Also x ul template DNA from a sample is added, where x depends on the total concentration of DNA in the sample. Typically 50 to 100 ng of total DNA is added. 7.5 - x ul of H2O is added to the mix. | Contents of the PCR mix is the for a large part the same as mentioned above for the Colony PCR. Differences will be noted here. First, instead of biobrick primer, any primer of choice can be added, also 2.5ul if standard solution has a concentration of 10 pmol/ul. Also x ul template DNA from a sample is added, where x depends on the total concentration of DNA in the sample. Typically 50 to 100 ng of total DNA is added. 7.5 - x ul of H2O is added to the mix. | ||
| Line 25: | Line 26: | ||
PCR program is: | PCR program is: | ||
1. 5' @ 95ºC | 1. 5' @ 95ºC | ||
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2. 1' @ 95ºC | 2. 1' @ 95ºC | ||
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3. 1' @ annealing temperature of the primer | 3. 1' @ annealing temperature of the primer | ||
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4. 1' @ 72ºC (1' is long enough for 1kb, longer times can be used if larger products are formed) | 4. 1' @ 72ºC (1' is long enough for 1kb, longer times can be used if larger products are formed) | ||
| + | |||
5. repeat steps 2-4 29x (total of 30 cycles, more can be added if necessary) | 5. repeat steps 2-4 29x (total of 30 cycles, more can be added if necessary) | ||
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6. 5' @ 72ºC | 6. 5' @ 72ºC | ||
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7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on) | 7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on) | ||
Latest revision as of 21:37, 21 September 2011
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Protocols
Colony PCR
1. Make biobrick mastermix, containing per sample
- 12.5 ul Taq mastermix
- 2.5 ul 10x forward biobrick primer
- 2.5 ul 10x reverse biobrick primer
- 7.5 ul H2O
2. Put 25 ul in the PCR tubes.
3. With a toothpick or pipet point, touch a colony and stir it through the fluid
4. Run the iGEM colpcr program (to be added later)
PCR using Taq Mastermix
Contents of the PCR mix is the for a large part the same as mentioned above for the Colony PCR. Differences will be noted here. First, instead of biobrick primer, any primer of choice can be added, also 2.5ul if standard solution has a concentration of 10 pmol/ul. Also x ul template DNA from a sample is added, where x depends on the total concentration of DNA in the sample. Typically 50 to 100 ng of total DNA is added. 7.5 - x ul of H2O is added to the mix.
PCR program is: 1. 5' @ 95ºC
2. 1' @ 95ºC
3. 1' @ annealing temperature of the primer
4. 1' @ 72ºC (1' is long enough for 1kb, longer times can be used if larger products are formed)
5. repeat steps 2-4 29x (total of 30 cycles, more can be added if necessary)
6. 5' @ 72ºC
7. ∞ @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)
