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I hate my Klenow!


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9 replies to this topic

#1 dldavide

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Posted 21 August 2009 - 07:29 AM

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
95C/5'
slowly cool down to r.t.

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

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

#2 swanny

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Posted 23 August 2009 - 04:55 PM

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.
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#3 dldavide

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Posted 23 August 2009 - 11:33 PM

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.

#4 swanny

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Posted 24 August 2009 - 05:25 PM

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.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#5 dldavide

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Posted 31 August 2009 - 03:52 AM

[/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 75C) 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

#6 dldavide

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Posted 31 August 2009 - 03:53 AM

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

#7 RachelP

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Posted 02 September 2009 - 06:03 PM

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?

#8 dldavide

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Posted 02 September 2009 - 11:46 PM

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 37C 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

#9 RachelP

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Posted 03 September 2009 - 02:22 PM

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?

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 37C 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



#10 dldavide

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Posted 04 September 2009 - 03:22 AM

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




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