PCR problems when amplifying sequence from gDNA - amplification of ~1.2 kb from gDNA (May/24/2007 )

Hi all,

I'm having problems amplifying a ~1.2 kb fragment from a gDNA sample, I keep getting smears and i've done this reaction before and it's worked, but for some reason, it won't work now...

My reaction mixture is 50 ul in total:

5 ul of 10X Amp buffer
1 ul of MgSO4
1.5 ul dNTPs
1.5 ul rev primer
1.5 ul fwd primer
1 ul gDNA (10 ng/ul)
1 ul (1 U) pfx pol
37.5 ul millQ water

I'm doing a gradient PCR where the Tm goes from 48 degrees C to 30 and I'm running it on a 1 % TAE gel stained with SYBR gold.

Please help, I don't know whats going wrong??

-natty

I assume you already exchanged all the solutions against new ones.

When the reaction worked: did you also use pfx polymerase back then? Or was it Taq? Depending on the polymerase the ideal temperature can differ. Try a broader gradient (I normally go from 50°C to 70°C).

Check your programm. For several weeks I couldn't run a proper standard PCR. I changed everything, yelled, begged - nothing worked. Then I found out that some idot had changed a setting in my PCR programm.

Good luck

Did you set up the reaction with 10 ng gDNA before??
To me, that's way too few. Maybe you should increase the amount of template DNA.
Ellis

Hi! I want to suggest you two ways to check out where the trouble is. Firstly, you had better to get a well-worked gDNA or some other templates to test whether the other gradients work. If you can get your target PCR product, we can make sure that it is the problem of your gDNA. If you can't get the target PCR product, I suggest you should use new agents or other gradients. Before this , you should make sure that your best PCR conditions is the same as before. Good luck!

If the PCR worked before and is not working now. Check all the different components for PCR. make new working solutions for dNTP's, primers, buffer etc...., even water.

hi

i have been following these tips and find them extremely useful

thanks every one for creating such a unique forum that goes a long way in removing the mystry from molecular biology

i was trying to amplify a gene from gDNA for the past three months but did not succeed

finally we discovered that the ctab method was inhibiting pcr

so we ordered a genomic dna kit

we got pure dna first time and i got my pcr sharp band with 0.5 ul of g dna
2ul did not give any band and and 1ul gave faint band
my pre-amplified control gave me positive result
however when i repeated with 0.5 ul in all 4 the pooled and run gel i got only dimers

kindly help me

genecat, can it be contamination and your DNA just gone? or perhaps you was able to introduce PCR inhibitor? A repetitive experiment and you are not getting anything? Weird.