Technique / Molecular Biology / PCR / Genomic DNA PCR


genomic DNA PCR



genomic DNA PCR

> Can someone forward me a general protocol for doing PCR using genomic
> DNA as the template? I seem to recall needing about 100 ng of DNA, but
> I forget stuff like how much Taq to use, concentration of dNTP's, and
> concentration of primers.

General protocol for PCR
Each person uses a slightly different ratio of reagents.

For DNA amplicons up to 500 bp, use 25 uL reaction volumes as follows:

On ice add to thin-walled pcr tubes

0.125 uL Taq polymerase (1.25 U, Gibco)
2.5 uL of 10 x PCR buffer minus Mg (1x)
0.75 uL of 50 mM MgCl2 (1.5 mM)
x uL template DNA (25-50 ng genomic dna)
0.5 uL of 10 mM dNTP mix (0.2 mM)
1.25 uL 10 uM primer mix (0.5 uM)
water to 25 uL

If DNA amplicon is >500 bp, scale-up the reactions for 50 or 100 uL total
volume. Program thermocycler appropriately and perform PCR reactions. 100
ng genomic dna is roughly right , depending on complexity, quality etc.

Just wondering why increasing the volume would help for longer products?

The above protocol was pulled from a protocol where the total yield is
important, specifically to produce enough product for sequencing reactions.
Generally the yield drops a bit with longer products, so the rule of thumb
solution was to increase the total volume. These measures are simply to
conserve reagents.

Buffer
1.25u Taq per 50ul PCR (add last)
MgCl2 1.5mM
200uM dNTPs
0.5uM each primer
30ng human xsomal (10,000copies)
water to 50ul

Duncan

Last update 28-Nov-2000, Rating of 12 votes.

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By sahil on 20-Jan-2009
dear sir/madam
i have done the pcr,didn,t get the product.I want to ask the question, if the primer length is twenty mer then this length is sufficient for the pcr amplification
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By ratna on 28-Aug-2008
I have been doing PCR with cDNA from phage library. Initially my PCR worked with 2.5pM of primers (final concentration) though the yield was low. Now, there is no amplification though the reaction condition is still the same. Can anyone help me solve this? Plz suggest me what else can be done.
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By Sandya on 19-Aug-2008
Hi,
Can we use restriction digested genomic DNA as template for a normal PCR reaction?

Sandya
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By kumar on 21-Jul-2008
hi all
i have a question. can anyone please answer?

Can we use RAPD-PCR for studying polymorphism in insects belonging to a particular family or can it be used only to study the same species from different ecotypes?
thanks
Kumar
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By SM on 22-May-2008
RE 'dear sir/madam,

can you please help me with this basic question... as to why genomic DNA cannot be used as template to PCR amplify a particular gene using primers for that gene??'

This is not possible, as in most cases your gene of interest would contain intronic regions, which if your're for instance trying to clone your gene, would not be ideal for expression.
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By mahendra kumar verma on 30-Apr-2008
i am doing pcr from genomic DNA so please send me protocol for that
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By arup on 06-Jan-2008
dear sir/madam,

can you please help me with this basic question... as to why genomic DNA cannot be used as template to PCR amplify a particular gene using primers for that gene??
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By MADHU on 27-Dec-2007
dear friend,i am also doing same work but right now i didnt get primers information,if you know please sent to me inforamation.

thanking you,

madhu sudhan
research associate
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By nico on 11-Sep-2007
you should try this:
1) blast your primers,
2) test different [MgCl2] (from 1 to 4 mM final)
3) use longer annealing time (to promote denaturation of false pairing),
4) higher annealing temperature (to avoid false pairing)
5) longer polymerization time ( to leave polymerase more time to amplify : 1min/kbp)

Good luck and Enjoy PCR !!!
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By tamara on 15-Jan-2007
have some trouble with PCR just checked and the only difference I found was that I'm using MgSO4 instead of MgCl2 could this be the problem?

thanks
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By rakhee lohia on 04-Jan-2007
dear sir/madam ,
ihave done PCR using 100 ng DNA. but i am gettin smaller band size after amplification.my desired band size is a5oo bp. but i am geting approx 300 bp only.
shall i increase my extension time or i should do any modification in annealing temperature.
kindly help me out.
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By s.arulmani on 23-Nov-2005
dear sir/madam
i am working in Aequorea victoria GFP gene
what is the primer for amplification of the perticular GFP gene














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By amita on 09-Aug-2004
here i wanted to ask that with previously standardized conditions sometimes i get variable results

like some tiomes intensity of the product band is very low

sometimes product does not come at all when different strains used

plz reply what can b the possibility
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By Dee Bell on 28-Nov-2000
Actually you should optimize your DNA concentration and your MgCl2 concentration before running your experiment. Different polymerases and DNAs can amplify differently under different conditions. (did that sentence make sense?) For procedures like RAPDS and ISSRs:

I use 25ul reactions with the following:

2 - 4 mM MgCl2

10 - 100ng DNA

200uM ea dNTP

10 -20 picomoles primer

1 unit polymerase

2.5uL 10X polymerase buffer stock solution

add water to volume of 25ul


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