08 August 2014

DGE, Covaris, and DNA fragmentation

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4 -- 8 August notes

2014-08-04

• Major computer problems
• workstation shutdown over weekend while backing up. Did I start upgrades to new kernel? refuses to boot into operating system
• desktop freezes on wake-up from suspend in both 12.04 LTS with new kernel and with 14.04
• Observed Grace’s experimental ant colonies

ApGxL

RNA extraction. Started with 6 samples for Qiagen RNeasy kit but 4 of 6 (damn!) were lost when caps came off in Bullet Blender.

Grabbed 4 replacement ants from colonies downstairs as tests.

Also ran the RNAzol extractions that had RNA according to Qubit through RNeasy kit.

Sample	Method	ng/ul
04-B-HS	RNAzol + RNeasy	2.88
10-B-CT	RNAzol + RNeasy	0
22-B-CT	RNAzol + RNeasy	0
22-C-CT	RNAzol + RNeasy	0
15-B-HS	RNeasy	6.97
08-D-CT	RNeasy	6.84
J1	RNeasy	13.4
J2	RNeasy	14.7
J3	RNeasy	5.6
J4	RNeasy	0

• RNAzol + RNeasy losses too much RNA, use RNeasy only. Consistent with previous tests

Sent to Bioanalyzer.

2014-08-05

• Watched Grace’s experimental colonies and feed Pogo colonies

ApGxL

Ovation DGE System for 7 samples through SPIA protocol

Samples

05-B-HS 15-B-HS 08-D-CT J1 J2 J3 J4

2014-08-06

• Agenda for Aphaenophone

ApGxL

Talked to Scott about settings for Covaris DNA fragmentation.

For test samples (J1-J4), checked DNA concentration with SPIA amplified samples, prior to Post-SPIA protocol and purification.

Sample	ng/ul
J1	42.9
J2	52.0
J3	40.1
J4	38.1

• Looks great for all samples!

After Post-SPIA protocol, used Qiagen QIAquick PCR purification protocol for samples 1-5, and compared to RNAClean XP beads for samples 6-7.

Sample	ng/ul
05-B-HS	15.2
15-B-HS	26.9
08-D-CT	>60
J1	>60
J2	>60
J3	>60
J4	34.2

Re-quantified 10^-1 dilutions of J1, J2, J3

Sample	ng/ul	original (ng/ul)
J1	19.4	194
J2	21.5	215
J3	10.4	104

Great DNA in all samples! No obvious effect of different RNA purification methods.

Re-ran DGE sample from last week that was out of range

Samples	ng/ul	stock (ng/ul)
04-A-HS	5.83	58

Covaris DNA fragmentation

To optimize fragmentation, ran test with different run times

Made 4 aliquots of samples J1 and J2 at final concentration of 16 ng/ul (4 ul DNA in 46 ul TE).

Sample	Intensity	Duty Cycle	Cycles per burst	Treatment time (s)
J1-1	5	10%	200	100
J1-2	5	10%	200	110
J1-3	5	10%	200	120
J1-4	5	10%	200	140
J2-1	5	10%	200	100
J2-2	5	10%	200	110
J2-3	5	10%	200	120
J2-4	5	10%	200	130*

• Messed up settings on final run and the last 10 seconds were Intensity 0.1, Duty Cycle 0.5% and Cycles per burst 50.

Sent samples (and unfragmented cDNA) to Bioanalyzer).

Computing

Re-installed Ubuntu 14.04.

Essential progams:

• git
• LaTex
• Emacs
• R
• pandoc
• Meld

2014-08-07

Other things

• Workstation busted. Dropped off at Pine Computers for diagnostics.
• Completed review for MBE
• Painted and treated Grace’s experimental colonies T2-3 and T2-4
• made fresh methoprene solution: 2.5 ul in 500 ul acetone (5ug/ml concentration)
• T2-3: 8 ants each red (methoprene) and yellow (acetone control).
• T2-4: 21 ants each red (acetone control) and silver (methoprene).

2014-08-08

• Aphaenophone conference call

ApTranscriptome

Incorporated comments from NS. More revisions. Sent to SHC.

TODO:

• figure of HSP expression
• thermal safety margins
• tables with enriched gene sets

ApGxL

• J1-0, J2-0 and J3-0 are the un-fragmented DNA.
• Other samples are fragmented at different settings, from 100 to 130 seconds.
• Fragmentation worked well, actually quite similar for all times.
• Size about 200bp, as desired for library prep.

RNA extraction for 12 samples using RNeasy. Two-step elution, first 16 ul, second 8 ul for a total of 24 ul.

Qubit RNA assay results

Sample	ng/ul
07-A-CT	0
07-B-CT	3.4
11-C-CT	3.0
15-A-CT	2.6
20-C-CT	14.2
26-C-CT	7.4
07-A-HS	11.1
07-B-HS	17.2
11-C-HS	7.6
15-A-HS	6.4
20-C-HS	7.1
26-C-HS	12.0

• Observed Grace’s ants
• Project organization
• Computer stuff…jekyll not running on new installation
• Installed Beagle for Medicago