Tuesday 06/30/15

9:45 am

RNAi

  • setup RNase digestions
  • fixed cells (probably too late for these guys, actually started Tuesday night).

Primer design

  • would be useful to do by batch in matlab rather than manually
  • looking up existing matlab functions for getting gene sequences etc.
  • troubleshooting
    • getgenbank returns over a quarter million features when called on Drosophila abd-B. (32 Mb of sequences mapping to unassembled parts of the chromosome). actually it’s all of Chr3R, starting from heterochromatic bases
    • need to use the nucleotide search option in genbank, search for gene name and melanogaster, select transcript, get accession number of that.
  • exploring Jeff’s DesignPrimers wrapper for primer3.
  • installed primer3 (best to get the windows binary version)
  • Completely awesome (as expected). Could use a Tm choice feature?
  • primers designed for
    'Abd-B','NM_001275719';
    'abd-A','NM_169733';
    'Ubx','NM_169728';
    'en','NM_078976';
    'Antp','NM_206445';
    'Dfd','NM_057853';
    'lab','NM_001260024';
    'Pc','NM_079475';
    'Psc','NM_001299439';
    'Esc','NM_058083';
    'ph-p','NM_001297873';
    'ph-d','AM943679';
    'pho','NM_166827';
    'Sce','NM_058161';
    'Su(z)2','NM_079002';
    'Scm','NM_001260077';

Orders

  • ordered BG3-cl2 cells
  • ordered NEB sequencing kit (to replace elements I’ll use from JM/SW stocks)
  • ordered primer plate (all Fwd primers, then all rev primers)

To do

  • set up new RNAi experiments
  • design primers
Posted in Summaries | Comments Off on Tuesday 06/30/15

Monday 06/29/15

9:45 am – 5:15 pm

Testing RNAi knockdown

RNA isolation

(assuming you cultured in 6 well plates – scale accordingly)
* Wash cells in cold PBS (with Kc, might have to resuspend, spin down, wash in PBS, then spin down again)
* resuspend cell pellet in 350 ul of RLT Buffer (from QIagen RNeasy plus kit). Vortex for 30 seconds. I find that the sample is stable if stored at -80oC in this. You have to make sure you vortex before storage though.
* follow Qiagen manual for remaining steps to purify RNA, including the “gEliminator” columns to remove genomic DNA.

First strand cDNA synthesis

RNA dilutions

  • 5 ug of RNA (2.5 ug/uL) -> 2 uL RNA
  • 5 uL ddH2O (for 2 uL starting RNA) (40 uL)
  • 1 uL oligo-dT primer (9 uL)
  • 1 uL dNTP mix (9 uL)
  • heat to 65C for 5 min, return to ice.

per reaction (master)

  • 2 uL 10x buffer (18 uL)
  • 4 uL MgCl2 (36 uL)
  • 2 uL DTT (18 uL)
  • 1 uL RNase OUT (9 uL)
  • all samples with RT (1 uL per reaction)
  • all samples without RT

RNAi experiment progress

  • Cells treated with RNAi, Tuesday evening 6/23
  • Incubated 6/24-6/29. Fixed 6/29.
Posted in Summaries | Comments Off on Monday 06/29/15

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Wednesday 06/24/15

9:50 am – 8:00 pm

presentation prep

  • tuning talk and slides

Coding

  • rewriting Chromatin Cropper

New Organization

  • 100% of the action of the GUI should be executable as command line functions
  • (currently the main processing “steps” are still steps of the GUI that have the GUI specific structures passed around).

New approach to writing

  • write the thing first as a single script with a lot of separate code blocks for each main processing step
  • make each code-block into a stand alone function with default parameters
  • GUI just links buttons and pop-up menus (other pop-up GUIs, as in STORMfinder) with functions.
Posted in Summaries | Comments Off on Wednesday 06/24/15

Tuesday 06/23/15

9:40 am – 11:00 pm

Heat shock experiments

  • continue cell prep
    • LN2 treatment,
    • 5 min 1M HCl bath
    • RNase A digestion (30 min at 37C)
    • SSCT wash
    • de-ionizing fresh formamide
    • prepared new formamide buffers
    • prehybe and store at 4C
    • Bogdan will take from here

Probes to test on HS cells

  • 5 different sized yellow domains
  • B02 / B03
  • B05 – B07
  • B08 – B10
  • B10 – B12
  • L2D12 (385 kb)

Presentation

  • preparing slides for ASBMB meeting

MERFISH

  • looking at clustering with new pipeline on old data

Set up new RNAi

  • use cells grown for 2 days in complete Schneider’s with FBS
  • spin down, resuspend in serum free Schneider’s. (probably should do a PBS wash first next time).
  • New RNAi in replicate, on 6 well plates (20 uL each)
    • th, rho, water, Pc (v2), ESC, Pc (v2) + ESC + PSC + + SCM
  • incubating 50 minutes

Orders

  • order new S1 (somehow ran out!?): S1_A647, S2_A647, S3_A750
  • first strand synthesis kit arrived today
  • Zymo RNA prep kit arrived yesterday (Qiagen RNeasy kit still not arrived).
Posted in Summaries | Comments Off on Tuesday 06/23/15

Monday, 06/22/15

10:00 am – 5:15 pm 10:45 pm – 11:30 pm

Tasks

  • discussed with Dell again about memory (they make this unnecessarily complicated).

Experimental goals for today

  • check progress RNAi knockdowns
  • purify more RNA for RNAi
  • set up new RNAi knockdown with more controlled

Ordering

  • sent Alec quote from Dell
  • ordered Qiagen RNeasy kit recommended by SN
  • ordered superscript III first-strand synthesis kit
    • (might be able to borrow a few reactions from Jeff/Steven)
  • Biosearch probes for hb arrived today. Yay! (need to prep some embryos to test).

RNAi experiments

RNA cleanup

remove template DNA

  • first, DNase digestion of template following NEB protocol
  • “To remove template DNA, add 30 μl nuclease-free water to each 20 μl reaction, followed by 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.”

cleanup with Agincourt (Beckmann-Coulter) RNA-clean XP beads

  1. Add 1.8 volumes of beads to sample in 1.7 mL tubes (min sample volume 50 uL + 90 beads)
  2. Mix by pipetting, incubate 5-20 minutes at RT (not on magnet)
  3. Separate on magnet until clear
  4. discard solution
  5. rinse 3x in 70% ethanol (500 uL – 1 mL)
  6. let dry 10 minutes
  7. Elute in >30 uL RNase free water

Quality checks

  • RNA concentrations all in the ~1000 ng/uL – 1500 ng/uL range according to nanodrop
  • running on 5% (2 month expired) TBE Urea PAGE gel
  • also forgot to remove tape. Still getting substantial product trapped in wells. Both RNA and DNA ladder look smeary. (somewhat old DNA ladder, maybe it is also a bit degraded). Still, hard to explain the larger than band smears based on degradation when running a denaturing gel.

RNAi knockdown progress

  • all wells 100% confluent
  • no visible differences between wells yet

Cell culture

  • cells in Schneider’s + 10% FBS are not adhering
  • resuspend 11 mL of confluent cells in Schneider’s without FBS.
  • Add FBS to 4 mL of this mixture (400 uL to 4 mL). Plate cells on 75mm2 flasks and bring volume up to 15 mL complete Schneider’s (with 10% FBS)
  • RNA not ready. Resupended all cells in complete media
  • cells in complete media still not adhering. Hopefully they grow at least and we can spin-down when necessary.
  • plated cells on 18mm coverglass freshly coated with Poly-D lysine, (and UV sterilized) in 12-well plates (~2:45 pm). Low density but workable.

Heat shock experiments

  • heat shocked for 20 min at 36.5C the newly plated Kc cells (after about 1 hour to attach).
  • fixed cells at 36.5C.
  • prepping cells for DNA-FISH. Now in glycerol at 4C
Posted in Summaries | Comments Off on Monday, 06/22/15

Sunday 06/21/15

9:50 pm – 11:45 pm

RNAi

RNA prep

  • set up T7 reactions
  • 10 x 9 = 90 uL NTP buffer mix
  • 2 x 9 = 18 uL T7 pol mix
  • 2 x 9 = 18 uL RNasin
  • 6 x 9 = 54 uL ddH2O
  • add to 10 uL of template DNA per well.
  • incubating at 37 C for 10 hrs O/N

RNAi treatment

  • add RNAi in 12 well plates
  • 10 uL of ~800 ug/uL RNA samples from BB.
  • 750 uL of cells resupended in serum free Schneider’s media (from SFX)
  • setup (top left to bottom right)
    • Th (two wells)
    • rho (two wells)
    • water (two wells)
    • RNAs 1-3 (one well)
    • RNAs 5-6 (one well)
    • Pc (one well)
    • Psc (one well)

Cell culture

  • passaged cells to a new plate of SFX
  • moved some cells to a new plate of Schneider’s media + 10% FBS
Posted in Summaries | Comments Off on Sunday 06/21/15