PCR from genomic DNA - (Jul/05/2005 )

Hi all,

I am trying to generate a PCR product from pure genomic DNA (P:C and prot K treated) of 150 bp in length but I ma getting no where... Some suggestions would be great. Here is my reaction conditions:

94------ 5min
94---- 1min
53---- 30sec
74---- 30sec
74----- 5min
15----- infinite (or until I feel like seeing a negative result )

25 cycles, Vent polymerase (NEB), 100pmol primers

Primers are approx 20bp in length and have a Tm of 54 (both)

I have a feeling I'm adding to much DNA (500ng) as a template

Thanks

-phattysbox-

That is *way* too much template dna. Dilute by 100x at least. I would initially denature at 96. Your extension temperature sounds high. This could be a problem if your template has high AT content. If your primers have that low a Tm, then you likely are working with a high AT organism. I would lower the annealing temperature (or use a gradient PCR machine) or redesign my primers for higher Tm. You could have problems with your primer choice. Give us the organism, expected product, and primer sequences, and we might be able to help more.

-phage434-

You could also try doing a MgCl2 series. I would also try decreasing the annealing temp by about 2 degrees.

-Jada-

Hi phattysbox,
You cycler number seems to be insufficient, try 35 to 40 cycles.
What is your MaCl2 concentration?

Regards
Hadrian

-Hadrian-

QUOTE (phattysbox @ Jul 5 2005, 06:44 PM)

)

25 cycles, Vent polymerase (NEB), 100pmol primers

Primers are approx 20bp in length and have a Tm of 54 (both)

I have a feeling I'm adding to much DNA (500ng) as a template

Thanks

yeah, 500ng is on the high end, too much starting gDNA will cause inhibition.

Drop your primer concentration to 25 pmol unless your pcr reaction is 100ul or over 25-50 pmol should be enough.

Drop your annealing temp to 50C. A good rule of thumb is start optimization with the annealing temp at 5C lower than your primers
melting temp.

Hadrian makes a good point, increase your cycle numbers to 35-40

-dobbiewalton-

Thanks fellas!!

I am going to try several ways to get my PCR product.

1) Dilution series of template gDNA

2) Dilution series of primer amount

3) Increase cycle numberto 35-40 cycles (good suggestion)

optimize, optimize, optimize...

By the way, I use Vent polymerase provided by NEB, which comes with own buffer and MgSO4 vails... I believe my MgSO4 concentration is at 1mM

-phattysbox-

You can also try to use a longer extension time i.e 1min at 72 degrees instead of 30 sec.

I have previousely tried amplifying a 1.6kb fragment from genomic DNA with 2 min extension but didn't work. When I increase the time to 3min I got my fragment.

-Muly-