I have some samples that were prepared and sent out to a sequencing facility to originally be run on an Illumina HiSeq, but through some unfortunate events these samples were not run on the HiSeq.
However, we do have a MiSeq at our institution and we want to run some of these samples that were originally prepared to be run on the HiSeq to now be run on the MiSeq.
The sample I want to start with is an 1000 ul enrinched library and is diluted down to 6 pM (I believe the sample is in the Illumina HT1 buffer + 0.1 N NaOH). The sample has been sitting in the -80C for about 6 months or so. I am trying to figure out what would be the best course of action to take in order to run this sample on the MiSeq and to have a successful run. Here are some of our thoughts so far:
- First we wanted to see if the sample was actually still at 6 pM by using qPCR and then continuing on with running the sample if it was still at this concentration.
- I also don’t know if the sample would still be denatured after sitting in the -80C for this period of time. So if we did want to qPCR the sample and move on would the sample still be denatured from the NaOH? And if so we are dealing with a much larger volume now of sample (1000 ul) because it is in the HT1 buffer and NaOH so we would have to figure out how to account for the volume change, added buffer, etc.
- I also called Illumina and explained the situation to them and their advice was to: ethanol precipitae the sample to get it out of the NaOH, then resuspend the sample in water, bioanalyze it, and then go from there. But of course since this is a weird situation they are not sure if this will work.
I know this is an odd situation but if anyone has any good ideas or advice it would be greatly appreciated! Thank you.
However, we do have a MiSeq at our institution and we want to run some of these samples that were originally prepared to be run on the HiSeq to now be run on the MiSeq.
The sample I want to start with is an 1000 ul enrinched library and is diluted down to 6 pM (I believe the sample is in the Illumina HT1 buffer + 0.1 N NaOH). The sample has been sitting in the -80C for about 6 months or so. I am trying to figure out what would be the best course of action to take in order to run this sample on the MiSeq and to have a successful run. Here are some of our thoughts so far:
- First we wanted to see if the sample was actually still at 6 pM by using qPCR and then continuing on with running the sample if it was still at this concentration.
- I also don’t know if the sample would still be denatured after sitting in the -80C for this period of time. So if we did want to qPCR the sample and move on would the sample still be denatured from the NaOH? And if so we are dealing with a much larger volume now of sample (1000 ul) because it is in the HT1 buffer and NaOH so we would have to figure out how to account for the volume change, added buffer, etc.
- I also called Illumina and explained the situation to them and their advice was to: ethanol precipitae the sample to get it out of the NaOH, then resuspend the sample in water, bioanalyze it, and then go from there. But of course since this is a weird situation they are not sure if this will work.
I know this is an odd situation but if anyone has any good ideas or advice it would be greatly appreciated! Thank you.
[The arrow thing, not precipitate 6pM DNA
]
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