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Team:DTU-Denmark/PCR program design
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(Created page with "== PCR program design == Step 1: Initial denaturation for 2 minutes at 95⁰C Step 2: denature for 1 minute at 95⁰C Step 3: Anneal primers for 30 seconds at temperature ~5⁰C ...") |
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== PCR program design == | == PCR program design == | ||
| - | + | # Initial denaturation for 2 minutes at 95⁰C | |
| - | + | # denature for 1 minute at 95⁰C | |
| - | + | # Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers. | |
| - | + | # Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on whether you are using a polymerase with proofreading capabilities. If in doubt see manufacturers instructions. | |
| - | + | # Repeat steps 2-4 for 25-30 cycles. | |
| - | + | # Final extension for 10 min at 72⁰C | |
| - | + | # Cooling down to 4⁰C (usually time is set as eternity so it can be stored in the machine until you can take it out) | |
Revision as of 14:42, 18 September 2011
PCR program design
- Initial denaturation for 2 minutes at 95⁰C
- denature for 1 minute at 95⁰C
- Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers.
- Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on whether you are using a polymerase with proofreading capabilities. If in doubt see manufacturers instructions.
- Repeat steps 2-4 for 25-30 cycles.
- Final extension for 10 min at 72⁰C
- Cooling down to 4⁰C (usually time is set as eternity so it can be stored in the machine until you can take it out)
