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PCR amplification


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5 replies to this topic

#1 chandrazee

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Posted 18 August 2004 - 12:42 AM

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

#2 CKM

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Posted 18 August 2004 - 02:41 AM

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

#3 ale

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Posted 18 August 2004 - 04:36 AM

hi Chandra...

have u checked if your genomic DNA is ok for amplification?, maybe a quick control with other primers (16S e.g.)
rgrds

#4 chandrazee

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Posted 18 August 2004 - 05:35 AM

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

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

#5 lyrezxl

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Posted 18 August 2004 - 07:01 PM

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.co...g_no_Bands.html

good luck!

#6 CKM

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Posted 19 August 2004 - 12:36 AM

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.

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? :o

Greetings
CKM




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