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Originally posted by patisev View PostI seem to have the same problem while constructing small RNA libraries for Solid sequencing, do you have any experience with this equipment? Since the fragments I should select are almost the same size as adaptor-adaptor fragments, we are having trouble collecting them from the gel. Do you think that using less adaptor could help me out too? Any experience with Invitrogen eGel for this kind of size selection? Thanks!
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@Captainentropy
In the first 5 test cassettes I had it happen with 2 out of 5 wells.
But with the first new cassette it happened only in one well.
I am using 2% Ethidium Bromide free cassettes and I definitely did not touch the well walls or bottom for sure. Hope this improves and I just had bad luck with the first ones.
Have you ever run a PCR product on the Pippin Prep without a purification step like columns or beads in between?
Your adapter dilution sound very promising.
I think I will try it again. Thanks!!!
If you start out with 100ng you probably are also dependent on the high sensitivity chips. Have you ever had problems running samples from the Pippin Prep on an Agilent? The elution buffer contains much more salt than they recommend for these chips.
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@dagmar. Yes, I've seen that happen with the well. Only once or twice maybe. It was probably just a malformed well allowing more buffer to flow into it since the buffer chamber is right next to it. If you're seeing that all the time then I'm not sure. Maybe poking a hole in the bottom of the well would cause that?
I try to use at least 100ng of ChIPed DNA if I can. If I have more then I use more. I've gone down to 1:50 dilution and got good library yield based on the Bioanalyzer trace. In fact for that one test it was slightly better than 1:30, but I'd say with low amounts of starting material 1:30 is fine. I have used 1 ug of starting DNA with 1:30 adapters and still got great library yield based on Bioanalyzer. We're going to try and optimize that step though, finding the optimal amount of adapters to add to 10, 50, 100, 500 ng, etc. of DNA.
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I was just looking at the TruSeq kit from Illumina. After ligation they do 2 AMpure beads purification and then a gel purification.
Seemed like overkill and I am a little concerned about loss of DNA.
Any thoughts?
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Hi Captainentropy,
I am glad to here you recommend the Pippin Prep. The few samples I just recently got to run were size selected very well but I still have the problem of loading my samples. Did you ever experience that the sample wells would refill with buffer before you could load your sample? Even if I would load my sample very quickly after emptying the sample well I would loose some of my sample do overflow. I do not want to take that risk but running only 2 to 3 samples on one cassette is very pricy too.
I ligate over night as well. I also tried using less adapters but this would always end up in almost no library at all. May I asked with how much DNA you start out with.
Thanks!!!
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If you have access to one, I recommend the Pippin Prep. The lab next door to me tested the Caliper XT recently but settled on and purchased the Pippin (which we both used in another lab before). Since the Pippin uses electroelution there is little loss of DNA in the range you are selecting for compared to the manual gel-excision method - you'll lose at least 50% of your library that way in addition to the possibility of cross-contamination.
However, after the elution step there is no need for purification of the DNA prior to the PCR, you will only lose DNA doing that. You can amplify directly from the eluted sample. What I do is I take the elution volume (I elute 200-600bp) and divide it into 36 uL aliquots and how ever many that is is how many PCR reactions I set up for that sample. Usually this is between 7-12. Doing this means you are able to maximize the amount of size-selected DNA in the PCR step resulting in more library. This also means you can get away with fewer amplification cycles thus reducing the number of potential PCR artifacts.
As for the OP question, the 124 bp peak is definitely amplified adapters. I've seen it many times. The Pippin (or CaliperXT) will reduce/eliminate this. After I switched to the Pippin I've only seen any adapter peak once, and it was small compared to my size-selected library. Also, reducing the adapter dilution ratio helps. I use 1:30. However, my lab is moving away from that and we are now going to try to optimize the dilution amount for each library. More work, but we'll have better libraries. Also, Illumina (I think it was them) now suggests using longer ligation times. I do overnight ligations anyway.
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adaptor-adaptor ligation problem with SOLiD small RNA library
Originally posted by pbluescript View PostYou should use less of the adapters that you ligated to your sample, not less enrichment primers. A 10:1 molar ratio at most, but I usually use less. If you have too many of them during your ligation reaction, they will end up ligating to each other and then that will end up amplifying in your enrichment PCR step. Even if you size selected with an agarose gel, you can still get some, especially if you used a razor to cut out bands. Since they are smaller than your sample+adapter, the adapter-only ligation products can dominate the reaction even if they are at a lower concentration than your sample to begin with.
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Originally posted by odile View PostOne alternative from the E-gel is the recovery gels from Lonza (flashgels). I tested them last week and they work great.
Can you do tight seperations like 120bp from 150bp and collect library ranges up to 300bp with it? On the Pippin prep low single nanogramms of DNA can be loeaded what about the minimum load of DNA of the Flashgels.
Thanks!
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Originally posted by cascoamarillo View PostThe Pippin Prep that we have is been used by other people in the department, not me. They also do RNA-seq, but I'm not sure what's the potencial separation-resolution of that machine (it's still a agarose separation system). For my stuff, where the adapters artifacts (75 bp) and the library (100 bp) are so closed, was not an option.
Hope it helps.
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Originally posted by niceday View Postto dagmar. Some different ratios are 1:1 for getting rid of 150bp and smaller. That might be useful if you don't mind taking a potential hit on the 150bp area of your library range. Alternatively 1.5:1. I'm not precisely sure of the size but it might get rid of some or all of your 120-130bp fragments.
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to dagmar. Some different ratios are 1:1 for getting rid of 150bp and smaller. That might be useful if you don't mind taking a potential hit on the 150bp area of your library range. Alternatively 1.5:1. I'm not precisely sure of the size but it might get rid of some or all of your 120-130bp fragments.
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One alternative from the E-gel is the recovery gels from Lonza (flashgels). I tested them last week and they work great.
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Originally posted by dagmar View PostThanks. I guess you just saved me time and effort.
I was planning on using the Pippin Prep next but the test runs were a little disappointing. I had problems loading samples because 2 out of 5 wells would refill with buffer and the yield from the ones that worked was very little.
Do you have any experience with the Pippin Prep? I will not get the chance to try the Caliper XP. I guess I will give the Pippin Prep a second chance so any further advice would be great.
Hope it helps.
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Originally posted by pbluescript View PostAh... that would be a problem. For getting separation resolution like that, the beads and even an agarose gel wouldn't be that great. You might want to try PAGE or, if you have the money, the Caliper XT.
The Caliper is capable of some very tight size separation.Originally posted by cascoamarillo View Postyeah, the PAGE is very effective for that sizes (although more time consume); I did it with my small RNA libraries and apparently I get rid off all the adapter-adapter artifacts.
Maybe the system Pippin Prep gives you another solution:
http://www.sagescience.com/
I was planning on using the Pippin Prep next but the test runs were a little disappointing. I had problems loading samples because 2 out of 5 wells would refill with buffer and the yield from the ones that worked was very little.
Do you have any experience with the Pippin Prep? I will not get the chance to try the Caliper XP. I guess I will give the Pippin Prep a second chance so any further advice would be great.
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