Gel Electrophoresis

To check DNA integrity, ran gel electrophoresis.

Gel-making protocol

1. Tape sides of gel mold and insert comb(s). For the integrity check of genomic DNA, used 12-well combs that had an additional well for ladder (1 comb per mold)

2. Melt one 50mL falcon tube of 1.5% SB agarose gel in beaker by microwaving in ~15s intervals, swirling intermittently to mix. Once fully melted, allow to cool until the beaker is comfortable to handle.

3. Add 2uL 10,000X gel red to melted agarose, swirling to mix, then pour agarose into gel mold.

4. Allow to cool until fully set and slightly opaque (~30 min).

   If using immediately , remove comb(s) and begin use (see next protocol). If storing, leave combs in and refrigerate in a sealed container.

Electrophoresis

1. If necessary, make aliquot of “dosed” loading dye, by mixing 1000uL 2X Loading Dye with 1uL 10,000X gel red.
2. Place gel, with comb and tape barriers removed, in electrophoresis chamber. Ensure it is fully covered by buffer, and wells are placed by the negative (black) node.
3. For each sample, mix 2uL “dosed” 2X Loading Dye to 2uL DNA.
4. Load into wells, taking care not to puncture gel or eject sample from well.
5. Load equal volume of Fast DNA Ladder in ladder well.
6. Run gel at 100-120 volts for 60min.

Gels

Gel 1

Gel 2

Gel 3

Notes:

• 10X = used 10X dilution of DNA to avoid high-concentration DNA “clogging.” Same dilutions as used for Qubit.

• 2nd = 2nd extraction of this specimen

• A few lanes were too faint to really see (1007393 10X, 1606824 10X, 51727, 51861), likely due to low DNA concentration. Can rerun 1007393 and 1606824 undiluted.

• Most of the old tuff (14366, 19054, 52295, 1180630, 50603, 14399, 42137, and 50368) seems ok. A few have HMW DNA, but most range from 1kb - 0.15kb. 42137 is the only one with many short fragments.

• Modern stuff also looks ok, especially the 1740### samples, which I believe correspond to the 2019 collections. These have lots of HMW DNA!

• Surprisingly, the 60s stuff seems most degraded (51857 - 51861, 51727). I may want to consider pulling different collections for this time range.

Reruns 7/22

Four of the samples had almost no visible DNA on the gel, despite having sufficiently high DNA concentrations (based on Qubit). Two were run as 10X dilutions, so I reran on a new gel using undiluted DNA. The other two may have been non-visible due to user error? e.g. maybe the DNA “floated” out of the well?

Samples to rerun:
USNM 1007393 (rerun undiluted)
USNM 1606824 (rerun undiluted)
USNM 51727
USNM 51861

Followed the same gel-making and electrophoresis protocols provided above.

Plot

library(googlesheets4)

Warning: package 'googlesheets4' was built under R version 4.2.3

library(tidyr)

Warning: package 'tidyr' was built under R version 4.2.3

library(ggplot2)

Warning: package 'ggplot2' was built under R version 4.2.3

conc <- read_sheet("https://docs.google.com/spreadsheets/d/1zDMy7AOCM8Ug85q4vr9CoMDxfF-zDJk1DnaxakSt1Hg/edit?usp=sharing")

! Using an auto-discovered, cached token.

  To suppress this message, modify your code or options to clearly consent to
  the use of a cached token.

  See gargle's "Non-interactive auth" vignette for more details:

  <https://gargle.r-lib.org/articles/non-interactive-auth.html>

ℹ The googlesheets4 package is using a cached token for 'kdurkin1@uw.edu'.

✔ Reading from "E_tourneforti".

✔ Range 'E_tourneforti_samples'.

# Pivot the concentration and gel size columns to long format
conc_long <- conc %>%
  pivot_longer(
    cols = c(Concentration1.ngul, Concentration2.ngul, Gel1.Size.kb, Gel2.Size.kb),
    names_to = c(".value", "Extraction.Number"),
    names_pattern = "(Concentration|Gel)\\s?(\\d)"
  )

conc_long$Gel <- factor(conc_long$Gel,
                            levels = c("none", "smear, 0.05", "short smear, 0.15", "smear, 0.15", "smear, 0.3", "smear, 0.5", "HMW, smear"),
                            ordered = TRUE)
conc_long$Collection.Age <- ifelse(conc_long$Year.Collected < 1920, "pre-1920",
                              ifelse(conc_long$Year.Collected > 1999, "Modern", "1960s-70s"))
conc_long$Collection.Age <- factor(conc_long$Collection.Age,
                              levels = c("pre-1920", "1960s-70s", "Modern"),
                              ordered = TRUE)

ggplot(conc_long, aes(x=Gel, y=Concentration, color=Collection.Age, shape=Extraction.Number)) +
  geom_jitter(size = 2, width = 0.25, height = 0) +
  geom_hline(yintercept = 10, color = 'black') +
  theme_minimal() +
  theme(axis.text.x = element_text(angle = 45, hjust = 1)) +
  xlab("Gel size range (kb)") +
  ylab("Concentration (ng/uL)")

Warning: Removed 30 rows containing missing values or values outside the scale range
(`geom_point()`).