Any experience with SPRIworks II for 454 libraries on the forum? How does it compare cost-wise with 454 reagents? And how are the results? Does it save you much time?
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We have just got the SPRIworks and prepared two libraries for 454. In general the instrument is great. Low maintenance and walk-away preparation. I haven't calculate precisely the cost yet but it should be in the same range or just a little bit more expensive per library than manual RL prep.
The preparation is indeed very easy. After nebulization and Qiagen purification there is only five minutes of SPRIworks prep. The instrument needs 3.5 hours and ends up with 50 ul of library ready for measurements with fluorometer. Lab time saving is high but our main goal is standardization as we have more people preparing libraries.
Regarding reagents one has to use 454 adapters which can be purchased with MIDs. But there is still needed fluorescent standard for quantification which is included in 454 Rapid Library kit. We will use it from older kits but I do not know what we will do later.
The results were very good. The library looked on the Bioanalyzer exactly as in the 454 protocol. What we have learned is that one should not use more than 500 ng of starting material. Better to use less. For one library we have used 660 ng (strange case anyway) and not all short fragments were removed.
The yield of libraries was very high. The sequencing results were as good as the manual preparation (sequenced on the same plate).
We are planning to use it also for SOLiD fragment libraries as soon as the kits are available. We do not have Illumina so no experience with SPRIworks I.
Cheers,
Marzanna -
Wow, thank you Marzanna, that is very helpful information.
I didn't realise that a SOLiD version was coming out, but that's good to know if it is.
Cheers!!Comment
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OK, well, we got one and it was installed last week. So I'll let you know how it goes ..Comment
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We prepared in a meantime a couple of SOLiD fragment libraries. They are not yet sequenced but from the bioanalyzer the fragments look very good. No primers left. The yield was very good.
According to the protocol one has to start with 1ug of DNA. We have tried also less (around 800 ng). Beckman support told us that with 200 ng will work but maybe the adapters should be diluted. They suggest a 1:10 dilution of the adapters for 200 ng.
There is a size choice of 150-250 bp or 150-350 bp. The bigger fragments look better and can be used for paired-end sequencing as well. I hope the sequencing will be fine.
The SOLiD kits are already available.
Cheers,
MarzannaComment
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