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Addition of A overhang for dummies?


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#1 KimWG

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Posted 22 November 2009 - 11:46 PM

Hi all,

I am trying to clone a 480-bp insert using an Invitrogen TA cloning kit. I need to gel elute the band of interest (I have to use a universal primer on my samples -- trying to get arthropod DNA from bat feces -- but the primer will get some bat bands of a different size, hence the need to gel-elute). PCR is with a regular old non-proofreading taq. So why worry about adding overhangs? According to the kit instructions, gel elution can remove the A overhangs, and I'm having enough trouble as it is; I really want to maximize my efficiency here.

Anyhoo, so I am trying to figure out how to add the A overhangs, but the more detailed protocols out there assume that you are using a proofreading polymerase, which I am not. The Invitrogen protocol suggested includes a phenol-chloroform extraction, which seems like overkill for my purposes, and other detailed protocols also include a number of purification steps to get rid of the Pfu (or whatever), which don't apply to me.

An older post in response to someone else with a similar problem said this: "Try running PCR product on 2% agarose gel and see your band of interest plus others, gel purify your band of interest, add A overhang by incubating with Taq and salts at 70C for 10 mins and then setup TA-cloning."

Great, totally ready to do it, but there is no suggestion of how much of what to set up. Do I basically set it up ("it" being the amount of buffer, taq, H20) as though it were for a PCR, but without the primer? How much of the PCR product should I use as a template? Does anyone have a specific "recipe" they can share? I am a novice at this stuff, so I probably don't know whatever it is that I should in order to not mess everything up.

Thanks
Kim

#2 phage434

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Posted 23 November 2009 - 05:15 AM

Pretend you are setting up a PCR reaction and leave out the primers. Do an extension (70C) for 10 minutes.

But the more likely problem is that you are trashing your PCR product with UV during the process of cutting your gel. Minimize exposure, use long wave UV (365 nm) or blue light (better).

#3 KimWG

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Posted 23 November 2009 - 08:52 AM

Pretend you are setting up a PCR reaction and leave out the primers. Do an extension (70C) for 10 minutes.

But the more likely problem is that you are trashing your PCR product with UV during the process of cutting your gel. Minimize exposure, use long wave UV (365 nm) or blue light (better).


Thanks -- I actually have yet to do the gel purification, I'm considering it because after transformation and picking colonies and running a PCR with M13 primers, I'm getting too many short fragments -- these could be bat sequences. I don't see these when I run a gel to check the product initially, so the amount is faint enough not to show up on a quick and dirty 1% minigel, but it's there enough to cause me problems later on.

When you set up the PCR, do you use the same amount of product as you would template in the initial PCR? Example: I usually set up a 25 ul reaction, consisting of:
- 2X master mix (taq, buffer, dNTP's etc): 12.5 ul
- sterile ddH20: 9.5 ul
- primer mix: 2 ul
- template: 1 ul

Presumably, I would substitute the pcr product for the template, and adjust the water up to make up for the volume lost by not using the primers? Since it's product from a pcr, would I use less template? Or would I use more, since the point is not to make more copies, but simply add the A overhangs?

Thanks so much for answering what must seem like a dumb question to a non-novice. Before October, the last time I had picked up a pipettor was 1997, so I'm stumbling around in the dark. (And unlike the bats I study, I have no echolocation to help me out!)

Meanwhile, I will have to lobby for a blue light transilluminator! I read on another thread here that adding guanosine to the buffer and gel can help prevent against UV damage, but the poster was using it with TAE buffer. Anyone out there try it with TBE? I imagine it would still work, but I am no chemist. Just an old fashioned field ecologist, in over her head!

#4 phage434

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Posted 23 November 2009 - 02:56 PM

You will not amplify the template DNA, so the amount out = the amount in. You probably want a relatively high concentration.

#5 swanny

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Posted 23 November 2009 - 03:06 PM

What have you done to optimise the PCR reaction to get rid of your presumed bat sequences?
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#6 bob1

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Posted 23 November 2009 - 03:59 PM

Just add 10x buffer to 1x, some Mg2+ to 1.5 mM, dATP to 200 uM, and a small amount of Taq to your purified PCR product in Tris or water. Incubate at 72 ˚C for 10 min.

It is best if you don't add dNTPs, though the Taq is supposed to only add A's onto the end of the sequence.

Edited by bob1, 23 November 2009 - 04:01 PM.


#7 KimWG

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Posted 23 November 2009 - 10:59 PM

What have you done to optimise the PCR reaction to get rid of your presumed bat sequences?


Not much -- I'm using universal primers with relatively low Tm's on an unknown number of imperfect templates-- I understand the suggestion but for the time being I'm making a conscious decision to run a PCR that allows some less specific binding as a trade-off to amplify a wide range of targets. Sometimes even bat targets. If bat DNA continues to be a problem, even after purification, then I will definitely do as you suggest...

#8 KimWG

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Posted 23 November 2009 - 11:00 PM

Just add 10x buffer to 1x, some Mg2+ to 1.5 mM, dATP to 200 uM, and a small amount of Taq to your purified PCR product in Tris or water. Incubate at 72 ˚C for 10 min.

It is best if you don't add dNTPs, though the Taq is supposed to only add A's onto the end of the sequence.


Thanks, phage and bob -- I used the recipes/suggestions you gave on a gel-eluted product that I set up for a ligation tonight -- hopefully it will work! Thanks!




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