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Old 12-04-2009, 09:03 AM   #1
athos
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Default Mate Pair Library Prep

Has anyone been using this? We've been trying to prepare some libraries with the Illumina kit, and are struggling to get enough DNA through at various stages of the process to do this successfully.

How much DNA are people starting with for this library prep? Is anyone getting as much as 200ng back to put into the circularisation reaction after starting with 10ug? And is anyone getting a decent amount of the final library recovered from the final gel size-selection step (after the PCR?). It's possible we're getting problems specifically with the recovery from the gel-extraction stage...

Also, we're using the Covaris sytem for both the fragmentation steps - is anyone else doing this? This works really nicely for obtaining 3kb fragments at the first stage, but I did worry about using it directly on the circularisation reaction for the second 400bp fragmentation step (i.e. without doing yet another column clean-up). Mainly because we've had problems with using a Covaris on DNA that wasn't just in water/TE alone...

Sorry for the long post, any hints or tips appreciated!
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Old 12-06-2009, 01:13 PM   #2
ScottC
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Hi,

I'd also be interested in hearing people's experiences with the mate pair preps.

Athos:

We can get 200ng back without too much trouble (for most samples) starting with 2-5ug of genomic DNA. We're using the current Covaris protocol without beads in the tubes. We're using the Qiagen gel extraction kits as per the standard protocol, except that we dissolve the gel slice without heating, but with constant agitation at room temperature (though I don't think heating needs to be avoided for 3kb fragments - it's just a routine now!).

What size band are you cutting from your gel before circularisation?

We often get next-to-nothing back from the PCR enrichment step, which is why I'd be interested to hear of other people's experiences.

I've found the Covaris to be OK for 3kb fragments, but not as good as for the 300-600bp we usually use for PE preps (but that's expected I suppose). We haven't found it much good for larger fragments, despite the claims from the company (which keep changing, actually!) and the purchase of a few thousand dollars worth of extra equipment! Are you using the 'standard' (well, current) Covaris protocol to produce your fragments?

Lastly, you can also try posting your question at the solexa google group and see if they have any solutions.
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Old 12-07-2009, 04:20 AM   #3
athos
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Hi, thanks for the reply.

We're finding that the standard Covaris protocol (special tube holder and tubes with beads) for the approx. 3kb fragments is working pretty well (large peak at the correct size on the bioanalyzer), and we're cutting 3-3.5kb from the gel. The recovery from the gel after end-repair and biotin-labelling hasn't been great though (there's a peak on the bioanalyzer, but not at a quantifiable level). So we're probably either having gel recovery problems, or we're not putting in as much DNA as we think we are! (although we're measuring this by picogreen, which is normally OK).

We also have seen almost next to nothing after the PCR, although we have recovered a very low concentration library (very low bump on bioanalyzer). We haven't tried sequencing this yet - do you have any results from sequencing these libraries?

Also, when you do the second fragmentation step after circularisation on the Covaris, are you cleaning up the reaction on a column first, or just proceeding 'as is'?

Many thanks!
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Old 12-10-2009, 07:25 PM   #4
ScottC
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Hi Athos,

We don't normally clean up the reaction before shearing it. Also, we haven't tried sequencing a very low concentration library.

You may be interested to know that just the other day Illumina released a new protocol for mate pair library preps. They say it should "increase protocol robustness, improve usability and generate higher quality and more diverse libraries".

You can download it from iCom.

Cheers,

Scott.
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Old 12-17-2009, 10:19 AM   #5
Hamid
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Hello Athos/Scott,

We recently introduced our Covaris miniTUBE-Blue consumable for the generation of 3kb DNA fragments with a sample mass range of 2-20ug in 200ul sample volume. Please take a look at the protocol and shearing data at http://covarisinc.com/pdf/pn_400069.pdf .

Thank you

Hamid

Last edited by Hamid; 12-17-2009 at 10:20 AM. Reason: forgot to sign.
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Old 08-30-2010, 07:09 AM   #6
tamar
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Default mate pair library prep

Hi,

I have been using Illumina's new protocol, and still have problems getting enough material for the ligation step. In the one case where I did get enough DNA (about 300ng), the rest of the protocol worked well. It feels like I'm loosing the DNA at the gel purification step.

I will appreciate any help with this problem!
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Old 09-22-2010, 09:08 AM   #7
Efortier
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Hi,

Is it possible to use the Fragmentase for the 2 fragmentation steps instead of Covaris? Anyone tried that?

Thanks!
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