Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol. After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday, ran a full library “prep scale” PCR.
| **REAGENT** | **SINGLE REACTION (μL)** | **x11** |
| Template | 40 | NA |
| ILL-HT1 (1μM) | 5 | 55 |
| ILL-BC# (1μM) | 5 | NA |
| NanoPure H2O | 5 | 55 |
| dNTPs (1mM) | 20 | 220 |
| ILL-LIB1 (10μM) | 2 | 22 |
| ILL-LIB2 (10μM) | 2 | 22 |
| 5x Q5 Reaction Buffer | 20 | 220 |
| Q5 DNA Polymerase | 1 | 11 |
| **TOTAL** | **100** | **550** |
Combined the following for PCR reactions:
-
55μL PCR master mix
-
40μL ligation mix
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5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.
| **SAMPLE** | **BARCODE** | **SEQUENCE** |
| Oly RAD 02 | 1 | CGTGAT |
| Oly RAD 03 | 2 | ACATCG |
| Oly RAD 04 | 3 | GCCTAA |
| Oly RAD 06 | 4 | TGGTCA |
| Oly RAD 07 | 5 | CACTGT |
| Oly RAD 08 | 6 | ATTGGC |
| Oly RAD 14 | 7 | GATCTG |
| Oly RAD 17 | 8 | TCAAGT |
| Oly RAD 23 | 9 | CTGATC |
| Oly RAD 30 | 10 | AAGCTA |
Cycling was performed on a PTC-200 (MJ Research) with a heated lid:
| **STEP** | **TEMP (C)** | **TIME (s)** |
| Initial Denaturation | * 98 | * 30 |
| 17 cycles | * 98 * 60 * 72 | * 5 * 20 * 10 |
After cycling, added 16μL of 6x loading dye to each sample.
Loaded 10μL of ladder on each of the two gels.
Results:
(https://raw.githubusercontent.com/sr320/LabDocs/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg)
(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_01.png)
(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_02.png)
(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_03.png)
(http://eagle.fish.washington.edu/Arabidopsis/20151113_gel_oly_RAD_prep_PCR_04.png)
Things looked fine. Excised the bands from each sample indicated by the green arrow. Before and after gel images show regions excised. Will purify the bands and quantify library yields.