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 | NA |
| ILL-BC# (1μM) | 5 | NA |
| NanoPure H2O | 5 | 55 |
| dNTPs (10mM) | 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:
50μL PCR master mix
40μL ligation mix
5μL of ILL-HT1 (1μM)
5μL of ILL-BC# (1μM) - The barcode number and the respective sample are listed below.
NOTE: Samples 02, 03, & 04 did not have 40μL of the ligation reaction left (only 32μL) due to additional usage in the test scale PCR yesterday. Supplemented those three reactions with 8μL of H2O to bring them to 100μL.
| 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 |
|
|
| 12 cycles |
|
|
After cycling, added 16μL of 6x loading dye to each sample.
Due to limitations in available comb sizes and inability to combine combs to make larger well sizes, only loaded 58μL of samples in each well on this gel. Will load remainder on a second gel and combine after PCR products are purified.
Results:
(https://raw.githubusercontent.com/sr320/LabDocs/master/protocols/Commercial_Protocols/ThermoFisher_OgeneRuler_DNA_Ladder_Mix_F100439.jpg)
(http://eagle.fish.washington.edu/Arabidopsis/20151013_gel_Oly_RAD_prep_scale_PCR.jpg)
Well, this is lame. There are absolutely no PCR products on this gel. In fact, this just looks like big smears of degraded DNA. I was expecting an amplicon of ~166bp to cut out of the gel. Based off of the test scale PCR from yesterday, everything should have been hunky dory. Not really sure what to think about this…