PCR amplification - PCR amplification (Aug/18/2004 )
hello forum users
i have a problem in the amplification of a genomic DNA obtained from Bt cotton for Cry 1Ac gene while this gene get esily amplified when i am using plasmid DNA of E.coli clone having Cry 1Ac gene . what may be the reason ...????????
plz suggest me.
thanking in anticipation
CHANDRA
Hi Chandra,
what exactly is your "problem in amplification"?
Do you have no amplification at all? Do you get other products?
Please describe your problem a little bit more detailed.
In general, you have to make sure that your initial denaturing step is long enough when PCRing genomic DNA. I'd suggest at least 10-15 min at 95 °C.
If you have problems with unspecific bands, try a hot start PCR or higher annealing temperatures.
Greetings
CKM
hi Chandra...
have u checked if your genomic DNA is ok for amplification?, maybe a quick control with other primers (16S e.g.)
rgrds
what exactly is your "problem in amplification"?
Do you have no amplification at all? Do you get other products?
Please describe your problem a little bit more detailed.
In general, you have to make sure that your initial denaturing step is long enough when PCRing genomic DNA. I'd suggest at least 10-15 min at 95 °C.
If you have problems with unspecific bands, try a hot start PCR or higher annealing temperatures.
Greetings
CKM
thanks CKM for kind suggestions
i am not getting any amplification at all
and amplificaction temp. profile is in this way
T1= 94* C for 3 min
T'1= 94* C for 45 sec.
T2= 56.6* C for 1 min
T3= 72* C for 1 min
T'3= 72* C for 7 min
total 35 cycle.
genomic DNA is ok having A260/280 = 1.82 and without any shearing.
in spite of these all i am not getting any amplification with plant genomic DNA while proper amplification occur with plasmid DNA having same gene.
waiting for a kind suggestions.
thanks
chandra
hi,
PCR from plant genomic DNA should be much more different from plasmid. sometimes, the anneal temperature will be 6 or 8C degrees lower than that of plasmid. there is no band, isn't it? so maybe u'd better adjust anneal temperature again. try 50C or lower.
anyway, there's a website talks much more about PCR troubleshooting.
http://www.midsci.com/docs/sharkcage/caged...g_no_Bands.html
good luck!
T'1= 94* C for 45 sec.
T2= 56.6* C for 1 min
T3= 72* C for 1 min
T'3= 72* C for 7 min
total 35 cycle.
Hi,
lyrezxl is right, you should try to lower your annealing temperature. Perhaps you have a cycler where you can set a gradient.
Also, your elongation step at 72 °C could perhaps be a bit longer. But this depends on the size of your gene and the kind of polymerase you use. Have a look at the recommendations for your polymerase.
Your primers are suitable for your genomic DNA? I mean, not that your primers are compatible to a plasmid sequence rather than the gene itself?
Greetings
CKM
Hi,
You might wanna checkout this place: www.pcrguru.com
They have some tools and the eBook. I guess you have to buy the eBook, but the tools I believe are free...