Etchevers:Notebook/Genomics of hNCC/2008/11/25

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Results from sonication trials on E11 mouse tissues

Since I set up a redirect to this entry, here is the photo of the sonication. To go on paging forward in the transcription factors in eye development project, click here and to move back to the last entry, here.

Image:Sonications_small.jpg

The majority of fragments migrate below 450 bp for the 5x pulses and below 300 for the 7x pulses, so will do 4x pulses for next try with the 10 eyes.

The arrowhead is, I believe, unfragmented or poorly fragmented, large DNA. It at least moves out of the well. They also get some filamentous stuff rising from the pellet, that sounds just like long chromatin to me. I suggested putting less tissue.

E11 = Theiler 18 = C15 so that's just perfect.

The estimations are as follows:

  • 106 cells / mg tissue = 0.5 to 3 μg input DNA
  • In Nelson et al., Nature Protocols 1, - 179 - 185 (2006), they start with 3 x 107 cells, which gives them enough to make 13.33 classic ChIP reactions with 4-5 μg antibody. So per ChIP reaction, they suggest 2.25 x 106 cells or a bit more than 2 mg tissue.
  • They use each ChIP to perform 80 x 10μL qPCR reactions. I have as a rule of thumb to use 2 ng DNA in a typical reaction, though others find that low. Then again qPCR works well over a wide range of input DNA, depending how many cycles. So let's say it's 2 ng, though it might well be 20. That would mean a total yield of 160 ng per ChIP, usually in an output volume of 20 μL. I think that is the same order of magnitude that Christelle had for her whole C16 embryo ChIP sent to Switzerland.
  • Nicolas and Adeline used a whole E11 embryonic head. I'd say that was more like 10 mg tissue. (They in fact used three embryos?). And E11 abdomen(s). It may be less. In that reference, an average E12 whole embryo weighs 90 mg.
  • Heather 11:15, 25 November 2008 (EST):

Showing CMFDA distribution in rabbit corneal sections

Last September 26th, we injected hNCC from either cephalic or trunk levels into the injured rabbit corneas of four rabbits.

Angelique finished sectioning about 20 4-7 μm sections into each middle area of the cornea (they had been cut in half more or less and cut edges embedded down). We are using the recently received anti-fluorescein-R-phycoerythrin to see if can convert old exhausted fluorescein into bright red fluorescence.

1. Deparaffinated sections (did this before I gave her my more progressive protocol):

10' at 60 °C and melted paraffin 2x 4’ xylene (or toluene or Histoclear) 2x 4’ 100% EtOH 5' in running water Passage in PBS

2. Glycine (0.15% w/v in PBS) 10 min.

3. Passage PBS.

4. Block non-specific antibody binding with 10% goat serum in PBS for 30’. 300 μL per slide without a coverslip, this is preferable but be careful about drying.

5. Can reduce background as well by using Image-it Fx (Invitrogen I36933): apply a couple drops for 30’ under Parafilm coverslip.

6. Rinse PBS.

7. Primary antibody dilution 1:200 in 10% serum/PBS (anti-fluo-RPE or IgG)

• Place 100μL per slide under glass/plastic coverslip in humid chamber. • Incubate 2 hours at room temperature (RT).

8. Rinse 5x PBS for 5’ each (at least).

9. Drain to nearly dry, and mount with ProLong Gold from Invitrogen/Molecular Probes with DAPI already in it (P-36931): warm to room temperature just before use and keep dessicated.

10. Leave to cure at 4ºC o/n then nail polish around coverslips.

  • Heather 07:12, 25 November 2008 (EST):

Negotiated a new sample of RAX antibody (cf. this entry ) with Millipore and aliquotted some more.

Embryo embedding

  • 2x de-"amnionated" and one embryo still with amnion on were fixed for a good 20h in Formoy (modified Carnoy's solution) at room temperature. Perhaps a bit much. One further nude embryo was fixed in 4% buffered paraformaldehyde/PBS.
  • I rinsed the latter in PBS for 5' then in water, and in 1/2 water, 1/2 70% (therefore, 35%) EtOH for 45 minutes.
  • Then 20 minutes in 50% EtOH (all of these are under agitation).
  • Then at least 15 minutes in 70% EtOH, probably more, until Florence launches the automatic paraffin impregnation overnight. The two cassettes are under the reference number 08-1554. Go down tomorrow to embed them.
  • Heather 10:02, 25 November 2008 (EST):
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