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!
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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