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PCR Amplification
From 2008.igem.org
(New page: ==PCR Protocol== '''Material:''' Primers (FWD, RVS) PCR Supermix Parent plasmid '''Method:''' 1. Measure the concentration of the primers and the parent plasmid as follow: Do a dilutio...) |
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==PCR Protocol== | ==PCR Protocol== | ||
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'''Material:''' | '''Material:''' | ||
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Primers (FWD, RVS) | Primers (FWD, RVS) | ||
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PCR Supermix | PCR Supermix | ||
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Parent plasmid | Parent plasmid | ||
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'''Method:''' | '''Method:''' | ||
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2. Mix 90ul of PCR supermix with 80ng of each DNA, 200nM of each primer | 2. Mix 90ul of PCR supermix with 80ng of each DNA, 200nM of each primer | ||
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3. Put 90µl in 0.5ml tubes. | 3. Put 90µl in 0.5ml tubes. | ||
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4. Find the length of the PCR product from Vector NTI | 4. Find the length of the PCR product from Vector NTI | ||
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5. For each Kb need 1 min for step 4/5 | 5. For each Kb need 1 min for step 4/5 | ||
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6. Run the PCR program | 6. Run the PCR program | ||
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7. Put finished tubes in -20 | 7. Put finished tubes in -20 | ||
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'''PCR program:''' | '''PCR program:''' | ||
CTRL Tube (controls temperature based on tube volume) | CTRL Tube (controls temperature based on tube volume) | ||
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LID = 100 | LID = 100 | ||
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WAIT AUTO (waits till lid is 100 before starting program) | WAIT AUTO (waits till lid is 100 before starting program) | ||
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1. temp{94} for 30 seconds. | 1. temp{94} for 30 seconds. | ||
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2. temp{94} for 30 seconds | 2. temp{94} for 30 seconds | ||
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3. temp{55} for 30 seconds | 3. temp{55} for 30 seconds | ||
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4. temp{72} for x min. (1min/kb) (68C for fragments > 5 kb) | 4. temp{72} for x min. (1min/kb) (68C for fragments > 5 kb) | ||
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a. perform 30 cycles of steps 2-4 | a. perform 30 cycles of steps 2-4 | ||
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5. temp(72) for x min. (1min/kb) (68C for fragments > 5 kb) | 5. temp(72) for x min. (1min/kb) (68C for fragments > 5 kb) | ||
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6. temp(10) forever | 6. temp(10) forever | ||
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All temperatures are in degree Celsius. | All temperatures are in degree Celsius. | ||
Revision as of 17:12, 28 October 2008
PCR Protocol
Material:
Primers (FWD, RVS)
PCR Supermix
Parent plasmid
Method:
1. Measure the concentration of the primers and the parent plasmid as follow: Do a dilution of 50:1 with 1EB buffer [ do 98ul tris into 2 ul of DNA ] Use the below formula to find the concentrations
Conc. (ng/ul) = OD reading * dilution factor * DNA factor = OD reading * 50 * 30
2. Mix 90ul of PCR supermix with 80ng of each DNA, 200nM of each primer
3. Put 90µl in 0.5ml tubes.
4. Find the length of the PCR product from Vector NTI
5. For each Kb need 1 min for step 4/5
6. Run the PCR program
7. Put finished tubes in -20
PCR program:
CTRL Tube (controls temperature based on tube volume)
LID = 100
WAIT AUTO (waits till lid is 100 before starting program)
1. temp{94} for 30 seconds.
2. temp{94} for 30 seconds
3. temp{55} for 30 seconds
4. temp{72} for x min. (1min/kb) (68C for fragments > 5 kb)
a. perform 30 cycles of steps 2-4
5. temp(72) for x min. (1min/kb) (68C for fragments > 5 kb)
6. temp(10) forever
All temperatures are in degree Celsius.
