Finished up the Hood Canal broodstock extractions and did some re-extractions of ones that I had either used up when making test PCR libraries or had come out degraded. Used the EZNA mollusc kit.
Ran out of tissue for SS2_5, SS3_18, SS3_19, SS2_3, SS3_20.
Quibit samples for concentration and updated sample sheet.
Did another set of 24 extractions using the EZNA Mollusc kit.
Qubit of extractions done on 10/21/15 and 10/28/15. Updated the sample sheet with concentrations.
Ran out gels for DNA extractions that had been done on 10/21/15 and 10/23/15.
Gel of DNA extraction done on 10/21/15.
DNA extraction done 10/23/15
Did another set of 24 extractions using the EZNA Mollusc kit.
Finished re-equilibrating the columns that were left in 1M HCL last night. I rinsed them 5 times with water by adding 700 uL of NFW to the column and centrifuging for 1 minute at 10,000g. Then I rinsed them 3 times with TE buffer. Then, following the optional column equilibration step in the EZNA kit manual I added 100 uL 3M NaOH at centrifuged at max speed for 1 minute.
Using these columns, I finished the DNA extraction that was started yesterday evening following the EZNA protocol. I then ran out the extraction on the DNeasy columns from 10/26 and the EZNA extractions with regenerated columns on a gel.
Gel pic of DNA extracted using DNeasy kit.
Gel of DNA extracted with EZNA kit using regenerated columns. Tissue was left in lysis buffer overnight.
As you can see, neither of them look very good. With the extraction using regenerated columns, I’m not sure if it was the columns or the fact that they were left overnight. At least the SS2 samples were all repeats, but the SS4 samples will likely have to be re-extracted. Just to be safe I won’t be leaving samples digesting overnight anymore or trying to regenerate columns.
The new EZNA kit came in in the afternoon, so I did a set of extractions using that kit and did not leave them overnight.
DNA Extractions for Common Garden Project
I ran out of columns in the EZNA Mollusc kit and the new one I ordered hadn’t come in yet. Since I’m trying to finish all of the South Sound and Hood Canal extractions before I leave on Sunday, I decided to do one set of extractions with the Qiagen DNeasy Kit. I’ve used this kit successfully for my population structure project. Annoyingly, I grabbed the wrong set of samples and ended up re-extracting ones I had done previously. I guess the more DNA, the better. These are notated with a B in the sample sheet. Used Qubit to get DNA concentrations.
Since today felt like a wasted day, I decided to set up some extractions to finish tomorrow morning. I was also out of Qiagen columns, however, so looked into regenerating some of the EZNA columns. Based on references here and here I added 500 uL of 1 M HCL to used EZNA columns and left them overnight. I made sure to only reuse columns that had previously been used for South Sound extractions just in case there is any DNA carry-over.
Left tissue in lysis buffer at 10:30pm at 56degC.
Common Garden Project
DNA extraction from Olympia oyster broodstock
Did not find NF2_8…
Olympia Oyster Population Structure Project
I started preparing genotype-by-sequencing libraries using the dried down samples from 10/21/15. Did an ApeKI digestion and ligation of adaptors.
Digestion:
| 1x | 50x | |
| NEB Buffer 3 | 2.μL | 100 |
| ApeKI | 1 μL | 50 |
| H2O | 17 μL | 850 |
Added 20 uL of digestion master mix to each of the 48 wells. Incubated for 2 hours at 75degC then held at 4degC until ligation master mix was ready.
| 1x | 51x | |
| T4 Ligase Buffer | 5 μL | 255 |
| T4 Ligase | 1.6 μL | 81.6 μL |
| H2O | 23.4 μL |
Added 30 uL of ligation mastermix to each well to give 50 uL total. Incubated at 19degC for 1 hour and 65degC for 20 minutes. Cooled to 0degC and put in freezer.
Common Garden Samples
Running low on EZNA kit columns so ordered another kit.
Phylogeography Project
Setup dry down of 200 ng DNA from 48 samples.
| BC1_12 |
| BC1_8 |
| BC2_7: 10.6 |
| BC2_13: 11.5 |
| BC2_9: 13 (8) |
| BC3_13: 108 |
| BC3_12: 15 (8) |
| BC4_2: 8.84 |
| BC4_17: 11.4 (8) |
| CA1_18: 24.4 |
| CA1_1: 36 |
| CA1_2: 17.4 (8) |
| CA2_9: 43.8 |
| CA2_12: 18.8 (5) |
| CA3_6: 62.3 (5) |
| CA4_1: 5.1 |
| CA4_7: 19.8 (8) |
| CA5_10: 48.8 (8) |
| CA6_8: 5.14 |
| CA6_15: 25.2 (7) |
| CA7_11: 7.4 |
| CA7_16: 19.1 |
| CA7_15: 14.9 (8) |
| OR1_ 1: 7.66 |
| OR1_5: 29.2 (7) |
| OR2_6: 12.9 |
| OR2_12: 15.8 |
| OR2_15: 18.2 (7) |
| OR3_15: 9.24 |
| OR3_20: 13.7 (7) |
| WA10_16: 25.4 |
| WA10_13: 9.52 (8) |
| WA11_10: 18.7 |
| WA11_22: 17.4 |
| WA11_4: 11.7 |
| WA11_17: 4.78 (8) |
| WA12_15: 24.4 |
| WA12_11: 4.18 (8) |
| WA13_12: 7.2 |
| WA13_5: 4.4 |
| WA13_15: 45.1 (9) |
| WA9_2: 11.4 (7) |
| WA1_16: 62 (8) |
| Conch_1: 12.4 |
| conch_2: 12.3 |
| conch_4: 9.65 |
| CA5_13: 21.8 (repeat with 2) |
| CA5_10 (repeat within) |
Keeping the calculations of uL to add on page 3 of this Google Sheet.
Set-up a MiSeq run with the 2b-rad library (concentration of .815 ng/uL) finished on Friday 10/16/15. I’m using the MiSeq in the Pritzker DNA Lab at the Field Museum and a v3 reagent kit. To prepare the library for sequencing, I followed Illumina’s instructions. These instructions 1st call for a 4nM library, which I prepared based off these instructions: CALCULATIONSANDCONVERSIONSINPREPARATIONFORIlluminaMiseqRUN. The fragments should be approximately 176bp, so I used the following formula to get the nM conversion factor: 10^6/660/N (where N is the size of the fragment in basepairs). Taken from this website. This formula gave 8.6 which I rounded to 8. Therefore, to dilute my library:
While preparing the library, Illumina gives options for the PhiX spike-in and the concentration to run on the flow cell. I chose a 2% PhiX spike in and 10pM as previous users of this MiSeq have reported overclustering at 11pM.
Common Garden 2b-RAD
Gel extracted 2b-rad PCR samples from Wednesday 10/14/15 using the Qiagen Gel Extraction Kit. Incubated 40 uL of elution buffer on the column for 5 minutes. Decided that going forward I would run PCR product out on a low melt gel and use gelase instead of the kit to save time/money.
Quantified gel extracted samples with High Sensitivity Qubit. Multiplied 37 uL by the lowest concentration to get ng per sample to add to pool, then calculated how many uL of each sample to add to pool. 37 * .373 = 13.8 ng.
| Population | Sample | ng/uL after gel | Vol to add to pool (uL) |
| Oyster Bay | BS_2_6 | 0.907 | 15.21499449 |
| Oyster Bay | BS_2_7 | 0.388 | 35.56701031 |
| Oyster Bay | BS_2_8 | 0.661 | 20.8774584 |
| Hood Canal | BS_1_5 | 0.731 | 18.87824897 |
| Hood Canal | BS_1_6 | 1.45 | 9.517241379 |
| Hood Canal | BS_1_7 | 0.853 | 16.17819461 |
| Hood Canal | BS_1_8 | 0.96 | 14.375 |
| Fidalgo | BS_1_4 | 0.873 | 15.80756014 |
| Fidalgo | BS_1_5 | 0.373 | 36.99731903 |
| Fidalgo | BS_1_6 | 1.07 | 12.89719626 |
GBS Population Structure
Made new annealed adaptors for GBS as the current stock was over 2 years old. Followed Buckler lab protocol. buckler_lab_genotyping_by_sequencing_protocol_20110808
DNA Extraction of Oly Broodstock
Primers
PCR for 2b-RAD MiSeq run
Used 10 samples from 9/28/15 digestion and ligation. Numbers in parentheses are so I know which tube from the ligation each sample is in.
Master Mix
|
1x
|
10.5x
|
|
|
NFW
|
23 uL
|
241.5
|
|
10 mM (each) dNTPS
|
2
|
21
|
|
10 uM ILL-Lib1
|
2
|
21
|
|
10 uM ILL-Lib2
|
2
|
21
|
|
5X Q5 buffer
|
20
|
210
|
|
Q5 Taq polymerase
|
1
|
10.5
|
Gel of 2b-RAD PCR product
I think I should make sure to mix in the EtBr when doing the post staining as that is likely why there are bright white sections on the gels. It was still much easier to see the bands though. Cut out the bands and left them in 1.5 mL tubes in 4degC for gel extraction on Friday.
Getting My Genes Wet 