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Old 11-30-2016, 10:16 AM   #3
Junior Member
Location: Moscow, Russia

Join Date: Nov 2016
Posts: 5

r.rosati, thanks a lot!

I should try your method of storing used chips ("the opposite procedure" here means 2x isopropanol -> 2x flush buffer-> 3x 100 ul of 50% annealing buffer, am I right?) Do you use such chips for initialization several times in a row, or only once?
There's almost never "a used chip from a run performed less than 2 days ago" in our lab, and I struggle a lot with the idea to just use a pristine new chip (once an error in preparing of pooled libraries was found out just before chip loading, so I prefer to open a new chip as late as possible). So you suppose that the main source of contamination from cleaning chip would be chlorite buffer traces, and chlorite destroys DNA? (I have another idea here, dubious, but still: what if I will perform the post-chlorite MQ cleaning not once, but twice and still use this MQ chip for initialization?)

My template preparation kit (if I understood the question properly) is Ion PI OT2 200 Kit v3 that I use for emulsion PCR and after this for enrichment of ISPs. There is no specific mentions in this kit's protocol about adding control ISPs to the sample, so I guess they must be already mixed with empty ISPs in the stock tube?
Before dilution and pooling of the libraries, I always check the lenght distribution of the libraries using Agilent 2100 Bioanalyzer, and usually it's a Gauss distribution with a peak about 200-280 bp. It corresponds to read lenght about 100-150 bp (after primers' and adapters' trimming).

Control ISPs that I add specifically before sequencing primer annealing (I guess they are TF_C) are indeed expired like most other reagents we have, I'm not sure about "very" (their expire date was 1-1.5 years ago). I wouldn't discuss why, that's just what we've got. I already asked our bioinformaticians about TF reading quality, but they couldn't interpret this, they just said that there's other methods to check the sample reading quality and these methods confirm that quality is OK.

As regards possible problems before the run: first, I often obtain less then necessary total RNA to start with (it's extracted from specific sources, like human leukocytes or single snail neuron, and it's impossible to get enough), but I guess that worst-case scenario here is decreased concentration of the libraries, and I dilute libraries manyfold anyway. I also may lose some rare transcripts, but I can't lose the whole library, can I?
Second, I am sometimes suspicious about the OT2 machine's performance, I even tried to export the run logs from OT2, but they didn't record to my flash drive. I consider to order Ion Sphere Quality Control Kit for Qubit, but this kit only measures template adapter/ISP adapter ratio and helps you to tell if there was no amplification, but it can't estimate possible contamination or fragmentation of amplified DNA. Are there any other ways to control OT2 function?
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