9 replies to this topic

[milla]

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.

[phage434]

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?

[milla]

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

[phage434]

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?

[milla]

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/µl and I used 1 or 2 µl.

[milla]

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

[vetticus3]

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.

V

[HomeBrew]

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.

[Adrian K]

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.

[milla]

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!