Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.
Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).
I ran PCR reactions on a subset (Sample #: 2, 3, 17, & 30) of the 10 samples that I performed adaptor ligations on 20151029.
PCR reactions were set up on ice in 0.5mL PCR tubes.
|REAGENT||SINGLE REACTION (μL)||x4.4|
|5x Q5 Reaction Buffer||4||17.6|
|Q5 DNA Polymerase||0.2||0.88|
Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.
Cycling was performed on a PTC-200 (MJ Research) with a heated lid:
|STEP||TEMP (C)||TIME (s)|
We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.
These samples were run on a 1x modified TAE 1.2% agarose gel (w/EtBr).
Looks like cycle 17 is the minimum cycle number with which we begin to see a consistent ~166bp band. Will continue on with the “prep-scale” PCR using 17 cycles.