Sample extraction: Day 2

Kaitlyn lead DNA extractions today while I bounced between lab, classes, and other obligations! She extracted DNA from the following samples:

Low pH:

• 1-T3
• 7-T2
• 8-T2
• 10-T3
• 3-T1

Ambient pH:

• UK-06
• 11-T4
• UK-05
• UK-08
• UK-02

Methods

Kailtyn and I followed this protocol. Here are some notes:

• Step 1: Kaitlyn carved out the tissue from histology blocks while I was in class! She scraped the tissue out for the first batch, so I know the technique between batches is consistent. She noted that some of the samples from this batch were shallower, so we may not be able to extract RNA from those samples.

Table 1. Mass of samples used for DNA extractions.

Sample ID	Mass (mg)
1-T3	19.7
7-T2	19.9
8-T2	19.8
10-T3	19.7
3-T1	20.2
UK-06	20.0
11-T4	19.8
UK-05	19.7
UK-08	20.0
UK-02	20.2

Figure 1. Histology blocks after carving out tissue.

• Step 9: Ethanol was evaporated out for 10 minutes
• Step 10: Kaitlyn noted we were low on Buffer TD1
• Step 15: For the first incubation-vortex set, the temperature was 56.9ºC instead of 56ºC. I loosened the foil and allowed some air flow, which brought the temperature to 56ºC.
• Step 17: Samples incubated an extra 3 minutes while waiting for the heat block to reach 80ºC
• Step 18: During the fourth incubation-vortex set, UK-O8 and (possibly) 3-T1 leaked.
• Step 23: Some of 8-T2 spilled before centrifuging the sample
• Step 27: Samples were placed in the fridge for quantification on Tuesday

Results

Today’s DNA concentrations were more consistent than batch 1. Summary information can be found in Table 1. The same information, along with phenotypic data, can be found in this file.

Table 1. Tissue mass (mg), DNA concentration (ng/µL), and total DNA eluted (ng) for each sample extracted. Treatments and extraction batches are also indicated.

Sample ID	Treatment	Extraction Batch	Tissue Mass (mg)	DNA Concentration (ng/µL)	Total DNA Eluted (ng)
2-T1	Low	1	19.7	31.2	1560
4-T3	Low	1	19.8	14.4	720
5-T3	Low	1	20.0	9.12	456
6-T1	Low	1	20.0	17.9	895
9-T2	Low	1	19.6	15.9	795
1-T3	Low	2	19.7	7.78	389
3-T1	Low	2	20.2	6.40	320
7-T2	Low	2	19.9	10.8	540
8-T2	Low	2	19.8	19.3	965
10-T3	Low	2	19.7	18.3	915
12-T6	Ambient	1	20.0	5.64	282
UK-01	Ambient	1	19.7	63.0	3150
UK-03	Ambient	1	19.8	11.6	580
UK-04	Ambient	1	20.0	9.26	463
UK-07	Ambient	1	19.9	6.40	320
11-T4	Ambient	2	19.8	15.7	785
UK-02	Ambient	2	20.2	10.5	525
UK-05	Ambient	2	19.7	9.26	463
UK-06	Ambient	2	20.0	16.0	800
UK-08	Ambient	2	20.0	7.86	393

Update 2018-10-15

Kaitlyn ran the samples on the bioanalyzer. The electropherograms, gel pictures, and results tables can be found in this folder. UK-05 had a funny pattern with multiple peaks, but two of those peaks were above 500 bp. The gel matrix in samples UK-04, UK-03, 4-T3, and 6-T1 were compromised, so the assay was unable to detect the upper marker in this assay. In both of these instances, I think I can still go ahead.

Going forward

1. Determine the appropriate bisulfite sequencing prep assay. Steven said I can start with this link.
2. Figure out if I can extract RNA from remaining samples