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|
|5x Q5 Reaction Buffer||20||220|
|Q5 DNA Polymerase||1||11|
Combined the following for PCR reactions:
- 55μL PCR master mix
- 40μL ligation mix
- 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.
|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)|
After cycling, added 16μL of 6x loading dye to each sample.
Loaded 10μL of ladder on each of the two gels.
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.