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PCR Amplification
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Revision as of 01:00, 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. For each Kb need 1 min for step 4/5
6. Run the PCR program
7. Put finished tubes in -20
PCR program:
(Based on the use of a PCR machine)
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.
