Tagged: Meara stichopi Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 16:28 on 2013/08/02 Permalink
    Tags: , , , , , , Meara stichopi, ,   

    Meara in situ for RNA binding proteins 

    Starting in situ with mixed stages of Meara. 15 min protk.

    ago a ago b boule bruno a
    gus a mago orb2 staufen

    03/08/13

    Added probes.

    05/08/13

    First day of washes. Some small juveniles have ripped tissues. I guess the 15min protk is too much for them, the rest looks ok.

    06/08/13

    Regular 5 PBT and 5 PTw antibody washes. Kept rocking in cold room.

    07/08/13

    Developing day. Stopped argonaute after ~3h, they were becoming too dark. Bruno, Staufen are clearly there. Boule is weaker, but something. The rest there is nothing. Putting new AP in cold room.

    08/08/13

    In addition to Argonaute I also stopped Bruno, Staufen and Orb2 before going to the collection trip. The rest are developing overnight a bit more (also included Orb2 to develop overnight.

     
  • Bruno Vellutini 11:26 on 2013/05/21 Permalink
    Tags: , , , , , Meara stichopi, , ,   

    Probe synthesis for Meara RNA binding proteins 

    Began the probe PCR for RNA binding proteins in Meara. Finished by May 24th.

    Gene ID ENZYME ng/µL
    Ms gus a BV226 T7 3409
    Ms boule BV229 SP6 501
    Ms staufen BV233 SP6 1141
    Ms ago a BV237 SP6 563
    Ms ago b BV238 SP6 1236
    Ms bru a BV242 SP6 1226
    Ms mago nashi BV244 SP6 336
    Ms orb2 BV247 T7 473

    Ms RNA binding

     
  • Bruno Vellutini 15:39 on 2013/04/17 Permalink
    Tags: Meara stichopi,   

    Meara miniprep check 

    Ran a PCR to check the miniprep swap I did last time. I selected 2 sequences that will be used for in situ probes and ran a PCR with primers from both genes (boule and gus a). The correct gene should be amplified by its primer pairs and confirm the identity of the miniprep. As it can be seen in the gel I did indeed swap the labels of the miniprep, so BV226 is gus a and BV229 is boule.

    2013-04-17 15.10.07

     
  • Bruno Vellutini 17:10 on 2013/03/25 Permalink
    Tags: , , , Meara stichopi, , , ,   

    Cloning of Terebratalia wnt2 and other Meara RNA binding proteins 

    Started a PCR with Terebratalia wnta, wnt2; and Meara ago a, ago b, ago c, bru a, bru b, mago, mex3, orb2.

    2013-03-26 11.47.59 2013-03-28 15.23.26

    12/04/13

    Put colonies to grow.

    Colony T7
    Ago a c1 BV235
    Ago a c2 BV236
    Ago a c3 BV237
    Ago b c1 BV238
    Ago b c2 BV239
    Ago b c4 BV240
    Bru a c1 BV241
    Bru a c2 BV242
    Bru a c3 BV243
    Mago c1 BV244
    Mago c2 BV245
    Mago c3 BV246
    Orb2 c1 BV247

     
  • Bruno Vellutini 14:36 on 2013/03/08 Permalink
    Tags: , , , , , , Meara stichopi, , ,   

    Cloning of Meara RNA binding proteins 

    Initial cloning of Meara RNA binding proteins. Selection: ago a, ago b, ago c, boule, bru a, bru b, gus a, mago, mex3, orb2, stau.

    10/03/13

    Only 3 genes had visible bands: boule, gus a, and stau:

    2013-03-10 17.12.53

    Ligated these overnight.

    11/03/13

    Transformation and plated the genes.

    12/03/13

    Colony PCR with 6 colonies from each:

    2013-03-12 16.58.53

    14/03/13

    Put colonies to grow.

    boule c1 BV226 BV229
    boule c2 BV227 BV230
    boule c3 BV228 BV231
    gus a c1 BV229 BV226
    gus a c2 BV230 BV227
    gus a c3 BV231 BV228
    stau c1 BV232
    stau c2 BV233
    stau c3 BV234

    15/03/13

    Extracted plasmid and did sequencing PCR for all using T7 primer. Dropped tubes into sequencing facility.

    26/03/13

    Checking sequences and they look good, but there is a problem! Minipreps BV226, 227, 228 aligned with gus a and not with boule!!! Check why…

    Screen Shot 2013-03-26 at 10.22.12 AM

     
  • Bruno Vellutini 12:22 on 2013/03/06 Permalink
    Tags: Meara stichopi,   

    Checked in new primers for Meara RNA binding proteins. Let the cloning begin!

     
  • Bruno Vellutini 22:43 on 2012/09/03 Permalink
    Tags: , Meara stichopi,   

    Today Andi, Aina, and me went on the boat to collect Novocrania anomala and Meara stichopi.

    N. anomala was collected by dredging rocky bottom and looking for specimens attached to rocks and shells. Rocks were kept in a large box with sea water and transferred to S4 tanks with flowing sea water at Sars.

    M. stichopi was also collected by dredging, but on mud bottom. Around 20 specimens were captured in a new spot: 60°14.946 N, 05°08.481 O

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel