Originally posted by molly burns
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Get rid of big fragments
I need to remove fragments > 500bp and already removed everything <100bp (I basically have a smear that starts around 100-150bp). I would like to try to use 0.7x (from bluescript gel) and keep the supernatant.
What would you do next? Clean-up the supernatant with Qiaquick or MinElute or add 1.8x beads and start all over again?
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Originally posted by odile View PostI need to remove fragments > 500bp and already removed everything <100bp (I basically have a smear that starts around 100-150bp). I would like to try to use 0.7x (from bluescript gel) and keep the supernatant.
What would you do next? Clean-up the supernatant with Qiaquick or MinElute or add 1.8x beads and start all over again?
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Originally posted by pbluescript View PostI have attached an image showing the effect of altering the ratio of AMPure beads/sample. You can see that using a lower ratio will remove increasing sizes of DNA fragments. You can even use this for size selection by using two different concentrations. For example, if you wanted to get a band centered ~400 bp, you could use 0.7X beads and then take the supernatant. This would include all the DNA fragments that were smaller than ~500bp and didn't bind to the beads. Then mix the supernatant with 0.9X beads and everything larger than ~300bp would bind. Elute the bound DNA and you'll have a fairly broad, but good enough to sequence, library.
If you try the beads, there can be a bit of lot-to-lot variability, so I would test them with a 100 bp library as I did in this gel.
I haven't used the Truseq kit, but I have used the Truseq adapters with outsourced enzymes/buffers and the same problem can occur.Do you know about Agilent Haloplex? Help me please!
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Originally posted by DavidAntonio View PostI am sorry about the Necro Post, but I have a problem in this moment about this. Could you tell me the weigth (in bp) of your 1 kb ladder? It would be very useful for my thesis. Thanks a lot.
The 1kb+ ladder in the image is this one:
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Originally posted by pbluescript View PostThe 100 bp ladder in the image is this one:
The 1kb+ ladder in the image is this one:
http://products.invitrogen.com/ivgn/product/10787026Do you know about Agilent Haloplex? Help me please!
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Originally posted by rnaeye View Postpbluescript,
what is the DNA ladder you used on this gel. thank you!
Take a look again at the text above each lane.
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Originally posted by Simone78 View PostI had the same problem(s). To get rid of the adaptor-adaptor dimers I now do size selection using 2% E-gel (great because you can also select more than one single fraction, so you have a backup in case something goes wrong during the pre-amplification step). And you don't need an additional purification step, just pipette out the water (containing your DNA) from the well. To remove the primer dimers I use AMPure XP (1.8:1 ratio) which removes (almost) 100% of the fragments <100 bp.
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Originally posted by pbluescript View PostI have attached an image showing the effect of altering the ratio of AMPure beads/sample. You can see that using a lower ratio will remove increasing sizes of DNA fragments. You can even use this for size selection by using two different concentrations. For example, if you wanted to get a band centered ~400 bp, you could use 0.7X beads and then take the supernatant. This would include all the DNA fragments that were smaller than ~500bp and didn't bind to the beads. Then mix the supernatant with 0.9X beads and everything larger than ~300bp would bind. Elute the bound DNA and you'll have a fairly broad, but good enough to sequence, library.
If you try the beads, there can be a bit of lot-to-lot variability, so I would test them with a 100 bp library as I did in this gel.
I haven't used the Truseq kit, but I have used the Truseq adapters with outsourced enzymes/buffers and the same problem can occur.
Great job! I played with this too. But my concern focused on the size adjusted concentration, your gel is very convincible.
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Originally posted by monad View Post2nd gel purification is the best option here, but you lose lots of library DNA. In this case, you can set additional round of PCR will take care of it. Stick with not more than 5 round of PCR. If your bioanalyer peak is good, you will be fine even with 10-15 cycles of PCR.
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Originally posted by pmiguel View PostI have a partial answer. We tried a few modifications to the TruSeq protocol, one of which was to do the PCR step on non-size selected library, followed by size selection on the Pippin Prep. Our yield of final library was much lower than what we got following the normal protocol. But it was still enough to sequence.
It worked.
But there are a couple of additional issues for a non-amplified library:
(1) The Y-adapters apparently cause library fragments to migrate aberrantly on some, but not all, electrophoretic conditions. Details are in another thread. I think they may run at their correct size on e-gels, but appear larger than they actually are under most commonly used electrophoretic conditions. After enrichment PCR the adapters are no longer Y-ed, so the effect disappears and you see the amplicon's true sizes.
(2) Just to state the obvious: when you work with very limiting amounts of DNA, you have to watch losses to binding against plastic-ware, etc. A typical microfuge tube may be able to bind 1 ng of DNA (complete guess.) So, if you have 200 ng, who cares. But if you have 1 ng total, you might lose the majority of your library if you don't use low-bind plastic-ware.
--
Phillip
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Originally posted by XIAOXIAO View PostHey if you used AmPure beads for purification, what is the ratio of library versus beads for purificaiton?
Best,
Simone
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