The AFLP Thread

[The Introvert]

Hey everyone, this is my first thread here. I didn't check to see if there was an obligatory intro subforum; if so, I'll do that later.

 

This thread is intended to as questions/get answers to all things AFLP. I'm currently working on a project for species delimitation and I have a few questions about some of the steps - I want to get stronger results than what I've currently been getting.

 

For the first question:

 

1.During the Restriction/Ligation portion of the AFLP process (hereafter referred to as R/L), what is the recommended incubation time after you've added your DNA?

 

Some sources have said ~2 hours @ 37C. What I've currently been doing is letting is sit for ~8 hours at room temperature (~23C). What are your thoughts on this? Is this a big deal? Could this alter my results?

 

2. What effects does freezing my reactions after the R/L process have on my results? Obviously if I'm letting them sit for over 8 hours, I'm not going to just chill in the lab until it's done; I have other things I need to do, and usually by the time it's done I don't have enough time to run a gel to see if I got a smear and start preselective amplification.

 

3. What would happen if instead of letting the reaction set at room temperature for 8 hours, I put it in the fridge (~4C) and let it set for a longer period of time? Would this work? How long would it have to be? I don't quite understand the rate of reaction for R/L step obviously, and I'm unsure how stable the DNA is at this temperature for extended periods of time.

 

That's it for right now! Any others please feel free to ask more questions and of course I would love to get answers...

 

-BW

[The Introvert]

No bites?

 

Perhaps this should have been posted in a different subforum?

 

Come on guys, help me out here!

 

-BW

[Arete]

No bites?

 

Now, now, only a handful of members here would know what an AFLP is, let alone be able to give you advice on them, so you'll need a little patience.

 

One issue is that species delimitation is something of a small niche in evolutionary biology, and another is that AFLPs are a somewhat antiquated molecular marker that few people in that small niche are actively using. One of the main reasons for that is that AFLPs are notoriously difficult to optimize - a set of protocols that work for one study almost never work well for another, so a lot of tweaking and fiddling are usually required to generate consistent results.

 

If I were advising someone embarking on a species delimitation study, I'd be strongly advising against AFLPs and pushing for a RAD tag approach instead. The reason would be threefold ). It's a newer method using next gen sequencing technology, so you get a lot more markers per dollar. 2) It's "sexier" so it will make for a more topical publication at the end - if you're aiming for Molecular Ecology or something similar. 3) The approach in terms of bench work is a lot more cookie cutter, so you'd have better data, easier and sooner.

 

What's the reason for leaving them on the bench? No incubator? R/L at non optimal temperature is likely to reduce yields and give smeary banding on your gels is what I would guess.

[CharonY]

Echoing what Arete said, I assume not many would even know what AFLP stands for. I also do feel that it is a bit old-fashioned and newer techniques have made it a bit obsolete. But to your question: assuming you avoid start activity 8h at RT should be fine, though I would aim to maintain the same time in case one has to start troubleshooting. Since the reaction should be in equilibrium further incubation at 4C should also not be too detrimental, that is always assuming no contamination.

The reason for higher temp and shorter time is usually that you can process the samples faster and reduce the risk of some accidents/contamination/whatever on the way. That being said, if there is a contamination 2h at 37C are likely to end as bad.

I would avoid freezing if you process the samples in a timely fashion, as freeze-thawing can reduce sample quality. I would set up a time plan that either utilizes the short protocol for immediate processing, or the o/n one with processing directly next day. If that is for some reason not possible, put it at 4C. But I would always recommend having a tight experimental plan in which samples are all processed quickly to save you time.

 

Edit: this is of course assuming relatively standard restriction enzymes and appropriate DNA:enzyme ratios etc.

Edited December 6, 2013 by CharonY

[The Introvert]

 

Now, now, only a handful of members here would know what an AFLP is, let alone be able to give you advice on them, so you'll need a little patience.

 

One issue is that species delimitation is something of a small niche in evolutionary biology, and another is that AFLPs are a somewhat antiquated molecular marker that few people in that small niche are actively using. One of the main reasons for that is that AFLPs are notoriously difficult to optimize - a set of protocols that work for one study almost never work well for another, so a lot of tweaking and fiddling are usually required to generate consistent results.

 

If I were advising someone embarking on a species delimitation study, I'd be strongly advising against AFLPs and pushing for a RAD tag approach instead. The reason would be threefold ). It's a newer method using next gen sequencing technology, so you get a lot more markers per dollar. 2) It's "sexier" so it will make for a more topical publication at the end - if you're aiming for Molecular Ecology or something similar. 3) The approach in terms of bench work is a lot more cookie cutter, so you'd have better data, easier and sooner.

 

What's the reason for leaving them on the bench? No incubator? R/L at non optimal temperature is likely to reduce yields and give smeary banding on your gels is what I would guess.

I've heard of RAD (read a paper or two that mention its existence) but haven't really looked into it as a viable option for this project, partially because my PI is insistent on AFLP (given the circumstances, it's understandable). The reason for leaving them on the bench? Unsure. It's what was in the protocol given to me - it seemed odd, and that's why I'm questioning it. Seems more logical to just incubate for the two hours; we have more than enough resources to do so.

 

As for people not knowing what AFLP stands for: sorry

 

As is obvious, that was my first post here. I didn't really do much digging, as far as the extent of others' knowedge here is concerned.

 

Echoing what Arete said, I assume not many would even know what AFLP stands for. I also do feel that it is a bit old-fashioned and newer techniques have made it a bit obsolete. But to your question: assuming you avoid start activity 8h at RT should be fine, though I would aim to maintain the same time in case one has to start troubleshooting. Since the reaction should be in equilibrium further incubation at 4C should also not be too detrimental, that is always assuming no contamination.

The reason for higher temp and shorter time is usually that you can process the samples faster and reduce the risk of some accidents/contamination/whatever on the way. That being said, if there is a contamination 2h at 37C are likely to end as bad.

I would avoid freezing if you process the samples in a timely fashion, as freeze-thawing can reduce sample quality. I would set up a time plan that either utilizes the short protocol for immediate processing, or the o/n one with processing directly next day. If that is for some reason not possible, put it at 4C. But I would always recommend having a tight experimental plan in which samples are all processed quickly to save you time.

 

Edit: this is of course assuming relatively standard restriction enzymes and appropriate DNA:enzyme ratios etc.

 

This makes sense. I initially thought that, for storage for times longer than a few hours, freezing would be the better option because even though DNA is relatively stable at 4C, some degradation could occur. I failed to consider that freezing and refreezing would proabably do more damage.

 

As for leaving the reactions on the bench: I'm going to forgo that method. The 2hr incubation seems like the much better option here. The issue with me is timing; I have a hectic schedule (I'm an undergraduate taking over a project for someone that left unexpectedly, so I'm learning on the fly) and I don't want to leave reactions in a incubator if there are other people (namely, professors and graduate students) that want to use it. Damn submissiveness (although probably rightfully so).

 

Thanks for the advice, both of you!

 

-BW

Edited December 7, 2013 by The Introvert

[CharonY]

With regards to shared lab equipment, it should be fairly easy to coordinate use (i.e. after x o'clock please take sample out and put into fridge). Communication if important for an efficient lab.

[The Introvert]

Another question here.

 

As previously mentioned, I'm taking over a project that's nearly completed.

 

I still can't get my final products have strong reactions; the data is there (after we run it through the sequencer) but it's weak, and all the peaks are under 100 (which is our cutoff for legitimate data). I noticed that in all the previous runs, ROX 799 was used: for mine, I've been using 509 because:

 

a) I was told to

b) 509 should be sufficient because there isn't any data thereafter (I think the number means the length of the run, temporally and physically)

 

My question is: could using a different marker (509 instead of 799) cause my reactions to show up differently? I don't know what else I could be doing wrong; I'm following protocol carefully and making sure I have reactions throughout via agarose gels.

 

On a side note, I noticed that the previous undergrad's gels for the R/L process are showing fuller smears than mine are. While typing this, I remembered that I had another question concerning the R/L process:

 

What difference would it make if I didn't allow the gemonic DNA to completely thaw before putting it into the reaction? Is it possible that doing so would result in lower concentrations of DNA in the samples? Additionally, should I vortex or centrifuge the samples prior to adding the DNA? They're diluted in a 1:19 H20 ratio so I guess it could be possible that different concentrations could exist throughout the test tube (Although DNA is water soluble so it should be even).

 

-BW