Hi Shuaichen,

If you're using Taq or an enzyme mix that contains Taq you will have an addition of an A residue at the 3' ends of your synthesised strands (ie a 3' overhang). One function of the end repair would be to remove this, leaving blunt ends.

Thank you Adam. I use Roche Long Template PCR System. The enzyme is a enzyme mix of two. But they don't tell what are they. 

Often when it is a mix it is a Taq and a proof reading enzyme. If that is the case some, but not all, PCR products will have a 3' over hang, so you should really consider the end repair.

How are you cleaning up you DNA after the end repair reaction? You could use a method that allows you minimise product loss and concentrate. One method I use is AMPure XP Beads.

Thanks Adam! Yes I use AMPure XP Beads too. Becase I just did this once, I'm not sure why I lost a lot of  samples after purification. What about your experience? I mean if you clean up 1ug of DNA, using beads, how much will you left after this?

Thanks!

Hello Shuaichen,

In my experience I tend to lose a lot more through the gel purification process than the AMPure XP bead clean up. Have you checked the concentration before the end repair and before the clean up?

Best Regards

Hi Max,

I checked the concentration before end repair. It was 77ng/ul. But I didn't check it right after end repair. Several other procedures were done after end repair. Then I checked the concentration again, it was 2ng/ul! So it's hard to tell in which step I lost most of my DNA. I'm just thinking whether it's the end repair.

Yes, I also lost a lot in gel purification. But don't have better ways to purify PCR product. I tried PCR purification. But my primer dimer is too many. It may not be the right way.

If you are doing gel purification to remove primer dimer you could could try using AMPure beads to remove the dimer. This should result in higher yields, but you will need to play around with the ratio of beads to template some. You could also try running multiple PCRs and then pool/concentrate them to increase your overall yield, assuming you have enough template to start with.

Best Regards

the last AMPure I did I got a 60% yield at the end (that was after an end repair reaction as well), but my DNA was in realtively large fragments (5 kb). As max says, you might be able to use the AMPure for the PCR purification, and eliminate the primer-dimer if you can get the bead concentration right for the PCR product size.

If you're not sure where you are losing DNA I'd make sure you check the concentration at each step.

Shuaichen,

When things like this happen, I just check everything on gels when I repeat the process. I routinely purify long DNA fragments with SPRI beads and haven't had any problems. Make 70-80% ethanol fresh for bead washes. How do you end repair? Using a kit or your own cocktail of enzymes? I definitely agree with Adam and Max. Check concentration by Qubit after bead cleanups to see where it is lost.

Hi Max

Yes, I want to remove primer dimer and some smear from the PCR product. I haven't used AMPure beads to purify PCR product. Is it expensive? And how do you know your purified DNA only have your aimed fragment but not any other ones? I mean if it's a smear in the gel, will you have more or less the similar longth product?

I upload a gel image of my PCR product, is this suitable for AMPure purification?

Thanks!

Hi Adam,

Thanks! Next time I will check concentration after each step.

Hi Kenneth,

Thanks for your reply. How long is your DNA fragment? For me, it's 8kb. I have AMPure beads in my lab but don't have SPRI. But if it really works fine, I can have a try.

By the way, did you get your long DNA fragment from PCR? Since for me, I PCR the 8kb fragment from genomic DNA. You can see from the gel I uploaded, each two adjacent lane is the same sample. I want to get more product in one reaction. If for you, when you can get the faint band and want to optimize to get more product in one run, what will you do?

Thanks again!

Hi Kenneth,

Forgot to say, I use NEB end repair module to do the end repair.

Hello Shuaichen,

AMPure beads are a little expensive but you can make your own for less cost if you really want to. I have not tried this before (although I have be meaning to one of these days) but I know others that have and they said they could not tell the difference and the cost is a fraction of AMPure beads. Here is a link to the White Lab protocol, on page 8 they give a step by step protocol of how to make your own:

http://static1.squarespace.com/static/5154a345e4b0feb1eb1819fc/t/52a7681ce4b0253945d2e24d/1386702876160/White+Lab+GBS+ddRAD.pdf

The size selection process is pretty straight forward you just need to adjust the ratio of beads to DNA. AMPure beads are greedy meaning that they bind more to larger fragment than smaller fragments, thus if you put in the right ratio of beads to DNA you can effectively remove smaller fragments (see attached picture for an example). This method is great for removing primer dimers or other small fragments without sacrificing yield. However, it is a bit more tedious, requires optimization, and if you are not making your own beads it may end up costing a little bit more.

Best Regards 

Hi Shuaichen,

Like Max just described, I make my own SPRI beads (same as commercial Ampure beads) as it is MUCH cheaper per sample if you are doing high throughput libraries.

Find a paper from 1995 by Trevor Hawkins I think. You just make PEG and salt solution by low heating overnight on hot plate, filter, and then mix with Speedbeads by Thermo.

Your product size should work fine with beads. I do 20kb sheared DNA routinely. It is also possible to pull down BAC and plasmid DNAs. 

Hi Max and Kenneth,

That home made AMPure prep seems great, I think I may consider switching! What sort of shelf life do you get from the homemade beads once prepared?

Kenneth, are the concs comparable, I use 0.5x AMPure beads, so I'm guessing 0.5x of the homemade beads?

If that protocol is correct (i'm sure it is), and you end up with 50 ml of bead prep from each ml of beads you buy, then switching is a no brainer in my eyes!