PCR Amplification

From 2008.igem.org

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==PCR  Protocol==
==PCR  Protocol==
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After primers are designed and the correct gene can be isolated, PCR is used to amplify the selected DNA for further use in our system.
 
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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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'''PCR program:'''
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CTRL Tube (controls temperature based on tube volume)
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LID = 100
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WAIT AUTO (waits till lid is 100 before starting program)
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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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'''Thermocycler program:'''
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4. temp{72} for x min. (1min/kb) (68C for fragments > 5 kb)
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Step1: 95C for 5 min.
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a. perform 30 cycles of steps 2-4
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Step2: 95C for 15 sec.
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5. temp(72) for x min. (1min/kb) (68C for fragments > 5 kb)
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Step3: [lowest primer annealing temperature] for 60 sec.
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6. temp(10) forever
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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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All temperatures are in degree Celsius.
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Step6: Hold at 4C.

Latest revision as of 00:56, 30 October 2008

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PRINCETON IGEM 2008

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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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