9 replies to this topic

[dldavide]

Hi everybody,

I'm trying to convert a ssDNA template (115nt, PAGE-purified) to dsDNA using klenow fragment and a complementary oligo (20nt, desalted). Urea-TBE-PAGE inspection of the product shows a pale band ca. 120nt long (expected product) and a nice, sharp and intense band of ca 400 nt!

How could it be?


Any comment is warly welcome


P.S.: Reaction Condition

Annealing
ssDNA Template 200 pmoles
Oligo 600 pmoles
TE buffer
95°C/5'
slowly cool down to r.t.

Extension
Klenow 5 EU
dNTPs  0.8mM fininal concentration
37°C/30'

I've also tried different annealing buffers (TE+NaCl, Klenow Buffer, A Buffer)...always the same result

[swanny]

Check your sequence for any areas of self-complementarity.

If you do have self-complementarity, you can generate a ~200 bp product, which will run ~400 nt if it is not denatured.

[dldavide]

Hi swanny,

thank you for your help. I'm dealing with a ssDNA template which comprises a random region (NNK20) of 60nt flanked by 2 constant regions up- and down-stream. I can exclude any self-paring between the constant regions, however I cannot rule out self-complementarity within the random region.

Although self-pairing is theoretically possible witihin the random region, I thought that running an Urea-TBE-PAGE would have shown only the ssDNA...

Have you ever experienced uncomplete denaturation of DNA samples during Urea-TBE-PAGE?

Thank you,
D.

[swanny]

dldavide, on Aug 24 2009, 05:33 PM, said:

Hi swanny,

thank you for your help. I'm dealing with a ssDNA template which comprises a random region (NNK20) of 60nt flanked by 2 constant regions up- and down-stream. I can exclude any self-paring between the constant regions, however I cannot rule out self-complementarity within the random region.

Although self-pairing is theoretically possible witihin the random region, I thought that running an Urea-TBE-PAGE would have shown only the ssDNA...

Have you ever experienced uncomplete denaturation of DNA samples during Urea-TBE-PAGE?

Thank you,
D.

Only if the urea was off.

What does the DNA look like on an agarose gel, say 3%? I know it won't be denatured, but you'll be able to discriminate between ssDNA and dsDNA. Run untreated ssDNA, ssDNA with primers and your final reaction.

[dldavide]

[/quote]
Only if the urea was off.

What does the DNA look like on an agarose gel, say 3%? I know it won't be denatured, but you'll be able to discriminate between ssDNA and dsDNA. Run untreated ssDNA, ssDNA with primers and your final reaction.
[/quote]

Hi,

i run a 2% high-resolution agarose gel (Sigma Aldrich - cat #) and still the same result. However, I tried also the same protocol but omitting the klenow inactivation step (15 min at 75°C) after the fill-in reaction and now I have a clear, sharp band of the expected length. I suppose that the dsDNA product is too short and undergoes denaturation during the inactivation step and subsequent reannealing which yields multmers...

Anyway thank you a lot for your help.

Cheers,
Davide

[dldavide]

if you are interested the high resolution agarose from sigma is cat #A4718. Quite surprising resolution for an agarose gel!

[RachelP]

I was just looking at your reaction conditions because I am about to use the Klenow for the same purpose- is the annealing step required? I was just planning to incubate with the Klenow and dNTPs. If I need the annealing step, I will have to use random primers. How long is your oligo?

[dldavide]

RachelP, on Sep 3 2009, 04:03 AM, said:

I was just looking at your reaction conditions because I am about to use the Klenow for the same purpose- is the annealing step required? I was just planning to incubate with the Klenow and dNTPs. If I need the annealing step, I will have to use random primers. How long is your oligo?

Hi,

I usually find convenient to perform the annealing step since it yields a clearer product without unspecific by-products. However, I have never carried out a klenow with random primers. As rule of thumb, annealing (heat and then slowly cool down) yields the formation of the most thermodynamically stable oligo-template pair and therefore is more reproducible. Conversely, incubating oligo+template at 37°C may lead to extension of kinetically favoured pairs and therefore could reduce reproducibility.

My oligo is 20nt long, whereas the template is 115nt long.

Ciao,
Davide

[RachelP]

Thanks, Davide. I noticed your oligo size in your original post after I asked. Do you think the conversion to dsDNA will work without the annealing step, or does the polymerase need the oligo to polymerize the second strand?

dldavide, on Sep 3 2009, 12:46 AM, said:

RachelP, on Sep 3 2009, 04:03 AM, said:

I was just looking at your reaction conditions because I am about to use the Klenow for the same purpose- is the annealing step required? I was just planning to incubate with the Klenow and dNTPs. If I need the annealing step, I will have to use random primers. How long is your oligo?

Hi,

I usually find convenient to perform the annealing step since it yields a clearer product without unspecific by-products. However, I have never carried out a klenow with random primers. As rule of thumb, annealing (heat and then slowly cool down) yields the formation of the most thermodynamically stable oligo-template pair and therefore is more reproducible. Conversely, incubating oligo+template at 37°C may lead to extension of kinetically favoured pairs and therefore could reduce reproducibility.

My oligo is 20nt long, whereas the template is 115nt long.

Ciao,
Davide

[dldavide]

Hi Rachel,

The synthesis of the complementary strand definetively requires an oligo to start the polymerization and more specifically a free 3'-OH. The annealing step is suggested in order to favour the oligo-template base pairing. How long is our template? How long is your oligo (or randomised oligos)?

Cheers,
Davide