PCR Amplification

From 2008.igem.org

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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
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5. Run the PCR program
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6. Run the PCR program
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7. Put finished tubes in -20
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'''Thermocycler program:'''
'''Thermocycler program:'''
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Step1: 95C for 5 min.
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CTRL Tube (controls temperature based on tube volume)
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Step2: 95C for 15 sec.
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Step3: [lowest primer annealing temperature] for 60 sec.
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LID = 100
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Step4: 68C for [1 minute + 1 minute per 1 kb).
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Go to Step 2: 35 times.
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WAIT AUTO (waits till lid is 100 before starting program)
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Step6: Hold at 4C.
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1. temp{94} for 30 seconds.
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2. temp{94} for 30 seconds
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3. temp{55} for 30 seconds
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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
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5. temp(72) for x min.  (1min/kb) (68C for fragments > 5 kb)
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6. temp(10) forever
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All temperatures are in degree Celsius.
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Revision as of 04:28, 29 October 2008

PCR Protocol

Materials:

Primers (FWD, RVS)

PCR Supermix

Parent plasmid


Methods:

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. Run the PCR program


Thermocycler program:

Step1: 95C for 5 min. Step2: 95C for 15 sec. Step3: [lowest primer annealing temperature] for 60 sec. Step4: 68C for [1 minute + 1 minute per 1 kb). Go to Step 2: 35 times. Step6: Hold at 4C.

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