Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-4-9
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ETOH Precipitation w/ tRNA Carrier + Library Construction Test[edit]
Background[edit]
- For a lot of the samples, the concentration is too low to fit into the Nextera reaction we already have established (we need ~1ng/2ul concentration). Because of this, we need to consider using ETOH precipitation in order to concentrate the samples to a level that's closer to what we need. I'm not going to use speedVac to concentrate the samples because the procedure is too variable for use.
- Consequently, today I'll be testing out the ETOH precipitation and library construction protocol. Because I've already made libraries for all the Catalina Harbor samples, I'll move on to make new samples from high input North Pond samples.
- The first part of the experiment will involve digesting the yeast tRNA (10mg/ml in 500ul, Life catalog #AM7119) using DNase I (from Richard, 10U/ul Roche catalog #04716728001) and then doing ethanol precipitation to ensure that all DNase is removed from the tRNA prior to adding with sample DNA
Procedure[edit]
Digest tRNA with DNase I[edit]
- Because we will need to use the tRNA for multiple samples, we will do many duplicates of this reaction and save the tRNA for later use
- I followed a similar protocol as provided by Roche to digest gDNA in RNA samples (see File:Cw 20150409 DnaseI protocol.pdf)
- Prep 0.2M EDTA (pH 8.0) from 0.5M EDTA by mixing: 20ul 0.5M EDTA + 30ul H2O. This will be used in the stop reaction
- Mastermix Recipe:
1x 10x tRNA (10ug/ul) 5 50 10x Buff 5 50 DNase I (10U) 1 10 Water 39 390 ---------------------------- Aliquot 50ul per reaction
- Incubate 37C 20min -> add 2ul 0.2M EDTA -> 75C 10min
ETOH precipitation of digested tRNA[edit]
- Combine 2rxn's per each non-stick tube
- Add 250ul 100% ETOH, 10ul 3M NaOAc, 1.5ul glycoblue