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 | * 98 | * 30 |
| 27 cycles | * 98 * 60 * 72 | * 5 * 20 * 10 |
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:
(https://raw.githubusercontent.com/sr320/LabDocs/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg)
[caption id=”” align=”alignnone” width=”701”]
(http://eagle.fish.washington.edu/Arabidopsis/20151112_gel_oly_RAD_test_scale_PCR.png) Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.[/caption]
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.