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PCR with genomic dna - (Aug/26/2009 )

Hi everybody!

I need some help with my pcr... I am trying to amplify a promoter (2kb) form human genomic dna.
The program that I am using is
95 x 5 min
35 cicli:
95 x 30 sec
65x 30 sec
12 x 2min
and 72 x 7 min

The problem is.... sometimes the pcr works well, sometimes so and so and sometimes i get nothing!

I am really carefully to do every time exactly the same, using the some reagent (pfx50 from invitrogen) and the same machine.

The region has a 39% GC content.

I will be grateful for any idea or suggestion that you can give me,

Thank you very much.


You really meant 72 x 2 min, correct?

You said nothing about your template concentration, or your template DNA preparation.
Have you tried lowering your annealing temperature?


No, actually I meant 72 for 7 min.
I have tried with 50 or 100 ng of dna.

The primer T is 64 and 67, I started with gradient pcr so I did lower and higher than 65.

What is shocking me is that sometimes I get such beautiful band!

Thank you


It's the line above that one, the one that reads 12 x 2 min that I was referring to. How are you preparing your genomic DNA? What volume of DNA do you add to your reaction as a template?


Yes, I am sorry.... is 72 x 2.

I get from the genomic DNA form biochain: is "Genomic DNA - Human Adult Normal Tissue: Heart, from a single donor". I made it at concentration =50ng/Ál and I used 1 or 2 Ál.


Please... can someone give me some suggestions???


milla on Sep 3 2009, 05:01 AM said:

Please... can someone give me some suggestions???

How big is the band you are expecting?
What is the recipe you use?
Where do you make up the recipe?
Is it a clean bench?
Do you keep everything on ice?

so on and so forth.



What kind of polymerase are you using? How did you calculate the Tm for your primers?

The first thing I'd tweak is to drop your annealing temperature a bit, perhaps to 62 degrees (but it depends on the Tm of your primers, and how you arrived at the Tm). Secondly, I might try extension at 68 degrees, and/or I might increase the extension time a bit (but both tweaks depend on what polymerase you're using. For example, you're right at the minimum for Taq at 72 degrees for 2 kb.). I would also drop the initial 95 degree step to 2 minutes; I only use 5 minutes for colony PCR.

If you can show us the primer sequences themselves, and explain your PCR conditions with a bit more detail, I'm sure we can provide more specific ideas.


Your Starting material is genomic DNA, your condition as below:
95 x 5 min
35 cicli:
95 x 30 sec
65x 30 sec
72 x 2min
and 72 x 7 min

Since you had done your gradient PCR and get your optimum at 65C.

My suggestion is
95 x 5 min
35< 95: 30 sec, 65: 40 sec, 72 x 2min 20 sec>
and 72 x 7 min

Try and tell me is there any improvement.
I suspect there could be a problem with your template DNA purity rather than the PCR condition. PRIMERS concentration could be a factor as well.
Attached File

-adrian kohsf-

Thank you to everybody for all your suggestions!

Meanwhile I did another PCR adding betaine and I get a beautiful band...I am going to clone it now to send it to sequencing.

I hope I get the right one!