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Problems regarding point mutations.... - (Mar/08/2013 )

I initially tried with 125ng primer concentration with Taq DNA polymerase PCR master to capture my annealing temperature to prevent chemical usage…Then I tried with 1.72ul (5picomole/ul), I also tried using 2.5ul of my primers (5picomole/ul)…In some PCRs I used DMSO to prevent dimers…atlast I tried using Emerald PCR premix with 3ul primers ( 3picomole/ul)…I have attached the pictures for your reference…Anyone out there plz help me to sort out the problem….
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-kartiga natarajan-

Try to clearly state the problem and question. I just see that you tried a lot of things and then show us a lot of slides. Summarize what works, what doesn't work, and ask a specific question. Companies charge $150 for mutagenesis. Is this something you really need to learn how to do?


I think the primers are not working...Is this correct? Or anyone think that the PCR works? I think the faint bands appear on the gel is the template i have used...My product size is 6.1Kb....Give me suggestions if anywhere i have went wrong....I have attached the details of my primers used and the details about my DNA marker...Thanks...
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-kartiga natarajan-

PCR on what?
Are you doing site-directed mutagenesis? On a 6.1kb vector?
Like a QuickChange protocol?

If so, running 18 cycles on a 6kb vector may be not even visible on gel, did you try to transform? The band you have is higher than 10kb, probably gDNA. If you want to see whether primers are working, you should increase cycles. But you can't use that reaction then for transformation, only for checking.


I wanted to change one aminoacid to another...From serine to aspartic acid...Im doing as per quick change mutatagenesis protocol...See the last two slides of my ppt and tell me whether my pcr have worked or not...Im using my wild type plasmid as my template....Thanks in advance..
Attached File

-kartiga natarajan-