PhD notebook

Updates from February, 2012 Toggle Comment Threads | Keyboard Shortcuts

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  Bruno Vellutini 18:00 on 2012/02/26 Permalink
  Tags: confocal ( 23 ), staining ( 5 )   

  Antibody staining for cyphonautes larvae 

  Execute the protocol for antibody staining with Chema to test in cyphonautes larvae of Membranipora. Chosen antibody is phospho-histone which binds to histone in chromatin revealing dividing cells.

  26/02/2012

  Day one of the standard protocol. Incubating overnight with rabbit primary antibody diluted 1:2500 in PTx+NGS.

  27/02/2012

  Day two, washes and incubation with secondary (rabbit) antibody (orange-red AlexaFluor 594) diluted 1:200 in PTx+NGS.

  28/02/2012

  Day three, regular washes and addition of Phallacidin with 1:1000 DAPI. Staining for 1h and mounting in glycerol.

  Mounted 3 slides. 1 is good, 2 has most embryos and most dirt, 3 has only a few embryos. Checked the slides in the fluorescent lamp: DAPI and Phallacidin look great and strong; Phospho-histone signal is widespread in the whole embryo, but seems to be slightly stronger in the apical region.

   
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  Bruno Vellutini 17:08 on 2012/02/26 Permalink
  Tags: in situ ( 43 ), maelstrom ( 15 ), nanos ( 25 ), piwi ( 32 ), pumilio ( 21 ), tudor ( 20 )   

  In situ for Membranipora NANOS, PIWIL2, TDRD1, PUM, MAEL 

  Samples

  • Mm Early: 2-128 cells + day 1 + day 3-4 + late cleavage
  • Mm Late: pre hatch + hatched + day 5 + day 7 + early and late cyphonautes

  26/02/2012 — Day 1

  Samples were put in 2 eppendorfs (early/late) and rehydration, PTw washes and first prehybe carried on there. So between washes there was a gap to wait the embryos to settle.

  On the last step, addition of hybe buffer, samples were aliquot to the following 24-well plate:

  	NANOS	PIWIL2	MAEL	PUM	TDRD1
  EARLY
  LATE

  Left overnight at 62 °C at 18h.

  27/02/2012 — Day 2

  Probes were already diluted at the stock concentration 50 ng/µL and I had to dilute MAEL, PUM, and TDRD1 to 1 ng/µL. Hybridization started at 11:30.

  29/02/2012 — Day 4

  Washes with a few modifications (due to busy day):

  • 60 min instead of 30 min at 25% hybe 75% 2X SSC (during Sars Seminar).
  • 3x 10 min 2X SSC at hybe temp.
  • PTw washes reduced to 5 min.
  • PBT washes reduced to 5 min.

  Anti-DIG put at 17:10, rocker at 4 °C.

  01/03/2012 — Day 5

  Regular washes.

  02/03/2012 — Day 6

  Developing started at 12:30 (~450 µL AP substrate solution).

  16:00 — Some signal seems to be appearing in MAEL. Early stages not so conclusive, but on late stages look like a bilateral pattern is emerging in the lateral flanks of the cyphonautes.

  03/03/2012

  16:00 — All five wells with late stages showing cyphonautes with the same bilateral pattern observed above.

  04/03/2012

  Whole day at 4 °C.

   
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  Bruno Vellutini 20:28 on 2012/02/21 Permalink
  Tags: biopython ( 4 ), blast ( 6 ), code ( 4 ), python ( 4 )   

  BLASTer script improvements 

  I made some improvements in the BLASTer script, described below.

  • To deal with some NCBI updates I changed the rettype and retmode of the Entrez.efetch command.
  • Since I am now trying candidate genes from other species I set the reciprocal BLAST to the respective organism database. Now each locus shows each reciprocal BLAST separately, when needed.
  • The loci frame and strand is now stored and printed along.
  • Major output formatting adjustments.

  An output example:

  Locus_978_Transcript_1/1_Confidence_1.000_Length_2580 | frame: +1 | candidates: DDX4, vas, vasa, pl10
  
  				gene                id                  accession           e-value
    Homo sapiens
  				DDX4                54514               NP_001160005.1      3.78e-141
  				DDX4                54514               NP_001136021.1      3.78e-141
  				DDX4                54514               NP_077726.1         3.78e-141 <<
  				DDX4                54514               NP_001160006.1      4.93e-141
  				DDX3X               1654                NP_001180346.1      7.40e-121
  				DDX3X               1654                NP_001347.3         7.40e-121
  				DDX3X               1654                NP_001180345.1      7.40e-121
    Danio rerio
  				vasa                30263               NP_571132.1         1.36e-146 <<
  				ddx3                566947              NP_001119895.1      3.05e-122
  				pl10                30116               NP_571016.2         1.16e-121 <<
  				ddx42               503932              NP_001032894.2      5.45e-79
  				ddx5                322206              NP_997777.1         4.32e-76
    Drosophila melanogaster
  				bel                 45826               NP_536783.1         8.20e-120
  				CG10077             38756               NP_648062.2         1.32e-77
  				CG6418              39218               NP_648413.1         2.11e-75
  				CG7878              40959               NP_649767.1         8.04e-75
  				Rm62                40739               NP_731034.1         2.19e-72
  				Rm62                40739               NP_731035.2         2.19e-72
  				Rm62                40739               NP_731033.1         2.19e-72
  				Rm62                40739               NP_001189182.1      2.19e-72
  				Rm62                40739               NP_731032.1         2.19e-72
  				Rm62                40739               NP_001163528.1      2.19e-72
  				Rm62                40739               NP_731031.1         2.19e-72
  				Rm62                40739               NP_524243.2         2.19e-72

  Now the next steps will be to integrate better with the Zim Desktop Wiki.

   
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  Bruno Vellutini 11:35 on 2012/02/19 Permalink  

  New germline genes 

  I selected more genes related to the germline development gathered from [1][2][3], etc. The current genes I am using are:

  vasa, ddx4, etc…

   

  10.1242/dev.00804

  10.1242/dev.047969

  10.1093/icb/icm027

   
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  Bruno Vellutini 08:00 on 2012/02/15 Permalink
  Tags: Priapulus caudatus ( 17 )   

  Field trip to Kristineberg marine station in Sweden to collect Priapulus caudatus.

  15-23 of February