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problem with pcr cloning from mouse cDNA


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#1 shenu

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Posted 05 June 2010 - 01:50 PM

Hi, I am new in molecular biology. I am trying clone some fragments from the cDNA. I designed the primers and first run my pcr. I got my desired products. Then, I tried to clone the same fragments from transgenic mouse ear clip samples with the same primers. I couldn't get any pcr products. I also tried with the same primer pairs with the transgene fragment in TOPO vector. Still I couldn't get any pcr product. If, any one can say why the problem arises. I am using herculase II pcr kit .

#2 blackmouse

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Posted 09 June 2010 - 12:28 AM

Hi shenu

the problem with PCR is it's still a mystery.
you have a whole bunch of options to optimize:

try different MgCl2 conc : 3mM or 5mM
try to run a temperature gradient on your pCR template as in 5-10C below optiomal to 3-5C above

the problem with amplifying from mouse ear (i have been through that just for genotyping) is that it is incredibly dirty
it depends on how you make your extract!!
Sometimes crude extract work better than the clean ones you get from a kit!!
yet crude extracts contain a lot of inhibiting factors!
so all depends on the template! I don't know what you use...
you can try and increase the primer conc. since you might titer it out if it binds to other stuff..try double it
same with nucleotides
beyond that make sure your primers are really specific by genome search

I had problems before observing all the above due to secondary structure to the template and adding DMSO helped...
not in lab right now so can't look it up but do a search on G/C rich templates and they give you a ball park for adding DMSO to the reacting and
it did the charm to me before.
good luck
let me know if you need more help
Cheers
A>

#3 shenu

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Posted 09 June 2010 - 01:49 PM

Thanks, i will perform the pcr again according to your suggestion.




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