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 |
| Template | 8 | NA |
| NanoPure H2O | 1 | 4.4 |
| dNTPs (1mM) | 4 | 17.6 |
| ILL-LIB1 (10μM) | 0.4 | 1.76 |
| ILL-LIB2 (10μM) | 0.4 | 1.76 |
| ILL-HT1 (1μM) | 1 | 4.4 |
| ILL-BC1 (1μM) | 1 | 4.4 |
| 5x Q5 Reaction Buffer | 4 | 17.6 |
| Q5 DNA Polymerase | 0.2 | 0.88 |
| TOTAL | 20 | 52.8 |
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) |
| Initial Denaturation |
|
|
| 27 cycles |
|
|
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).
Results:
Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.
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.
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