Search
Search
View additional product information for Oncomine Focus Assay, Chef-Ready Library - FAQs (A42008)
9 product FAQs found
After the completion of a run, the Ion Chef Instrument holds the barcoded libraries in the tube loaded in Position D of the Reagents cartridge. To avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as soon as possible after run completion. Do not leave the tube in the instrument longer than 24 hours after the start of the run. After 24 hours from the start of the run, the instrument chiller will stop actively cooling, and the sample will be held at 27 degrees C.
The tube in Position D of the Ion AmpliSeq Chef Reagents DL8 Cartridge will contain 700 µL of combined barcoded libraries at completion of the run. The libraries are at >>100 pM (total combined library concentration) and are ready to use in template preparation. One can store unused portions of combined libraries at 4 to 8 degrees C for up to 1 month. For longer-term storage, store at -30 to -10 degrees C.
Yes, fewer than 8 samples may be processed per run but keep in mind that a run consumes kit reagents for 8 samples regardless of the sample number.
If you are processing ≤5 samples, we recommend that you quantify your output combined library by qPCR to ensure that an optimal concentration is used in templating reactions.
If you are processing fewer than 8 samples, it is preferable to add replicates or positive control samples to the run. Otherwise, pipet 15 µL of nuclease-free water as non-template control into column 1 wells that do not contain a DNA sample.
The primer pools should be added to the Reagent Cartridge on the Ion Chef Instrument in positions A and B. Tubes in Positions C and D can be left empty. Uncap all 4 tubes in positions A, B, C, and D in the Ion AmpliSeq Chef Reagents DL8 cartridge.
Each reverse transcription reaction requires 10 ng of DNase-treated total RNA (≥1.43 ng/µL).
For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue.
8 samples can be processed per IonCode plate. The kit contains 4 IonCode plates, hence, a total of 32 samples can be processed using the Oncomine Focus Assay, Chef-Ready Library kit.
Reverse transcription needs to be performed manually in the IonCode 96-well PCR plate, using the SuperScript VILO cDNA Synthesis Kit. SuperScript reverse transcriptase enzyme is not supplied in the kit and must be purchased separately. If the RNA was prepared from FFPE tissue and not previously heat-treated, we recommend heating at 80 degrees C for 10 min, then cooling to room temperature.
The Oncomine Focus Assay, Chef-Ready Library contains the following components needed to prepare libraries for 32 samples:
- Oncomine Focus Assay, DNA Chef-Ready Panel (2X) - contains the DNA primer pool
- Oncomine Focus Assay, RNA Chef-Ready Panel (2X) - contains the RNA primer pool
- Ion AmpliSeq Kit for Chef Reagents DL8 cartridge - allows automation of the library prep on the Ion Chef Instrument
Note: The SuperScript transcriptase for cDNA synthesis is not included and must be purchased separately.