Monday 07/20/15

9:00 am – 8:00 pm

Goals

  • prep new RNAi treated cells for staining
  • Plate, fix and lyse current RNAi treated samples
  • RNA isolation prep
  • cDNA production
  • qPCR reactions (?) [tomorrow]
  • Set up synthesis reactions for new RNAi

RNAi experiments

new dsRNA synthesis

  • ran PCR cleanup on new samples of Pc-v2, Psc-v1, Esc, SCM (2 PCR tubes each / 100 uL total reaction)
  • set up T7 reactions:
    • 20 uL eluted DNA-template
    • 20 uL dNTP buffer mix
    • 4 uL T7
    • 4 uL RNasin
  • running for 16 hrs at 37 C

RNA isolation

  • following Qiagen RNAeasy kit
  • the second round elution does make a difference (1.25 – 2x more yield)
  • a third round elution does not help. temperature of water for elution does not matter (tried 4C and 37C)

sample order

  1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
  2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
  3. Pc v1
  4. Pc v2
  5. Pc, Esc, Scm,
  6. Esc
  7. Suz12
  8. Scm
  9. SCE
  10. mock
  11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

* concentrations in the 300 to 800 ng/uL range

First strand cDNA synthesis

RNA dilutions (master 1 12x)

  • (desired 10 ug of RNA) -> 20 uL RNA (have now ~200 ng/uL instead of 2500 ng/uL)
  • 2 uL oligo-dT primer (24 uL)
  • 2 uL dNTP mix (24 uL)
  • heat to 65C for 5 min, return to ice.

per reaction (master 2, 12x)

  • 4 uL 10x buffer (48 uL)
  • 8 uL MgCl2 (96 uL)
  • 4 uL DTT (48 uL)
  • 2 uL RNase OUT (24 uL)
  • 2 superscript3 (24 uL)

sample order

  1. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 1
  2. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 2
  3. Pc v1
  4. Pc v2
  5. Pc, Esc, Scm,
  6. Esc
  7. Suz12
  8. Scm
  9. SCE
  10. mock
  11. PPPES (Pc, Ph-p, ph-d, Esc, Scm) rep 0 (from last week)

final step

  • add 2 uL RnaseH to each reaction
  • I’ll set up the qPCR reactions tomorrow.
  • samples in -20C in front part of 8-strip rack.

Cell prep for staining

  • prepping all PPPES slides and wt controls for FISH
  • most of these slides have almost unusably low cell denisty
  • staining slide 1 PPPES (0) for Antp-P1 (2 uL + .8 uL S1) O/N.

Prep new cells

  • RNAi from 4 days ago ready to be fixed.
  • Plated 8 wells of new PPES (top for are PPPES rep-1, next for are biological replicate PPPES rep-2), + 2 WT/mock + 2 PES (Pc, Esc, Scm)

New dsRNA synthesis

  • Template making
  • PCR strip order:
  • Ph-p, Ph-D, Ez-v1, Ez-v2, Esc-v2, Psc-v2, Psc-v3, SCE-v2
  • running O/N

New RNAi knockdown

  • 3 wells of 6-well plate with 30 ng Ph-P and Ph-D
  • 3 wells of mock
  • not 100% sure I added the serum free media for the serum shock… Will see when we check the qPCR I guess.

RNAi planning

  • ph looks good, let’s push ahead with this as a single.
  • discussed doing RNA-DNA doubles. RNA with dig in conventional, antibody stained and post-fixed prior to digestion.
  • will try this soon — could use a P1-dig

Comparison of STORM data with Hi-C

  • some initial explorations
    HiC_comparison_Exponents HiC_comparison_InVsOut_bySpot HiC_comparison_InVsOut HiC_density_1 HiC_comparison_density_v2 HiC_comparison_density_v2bar HiC_comparison_density_v1

New Knockdown data

NewKnockdownStructureData

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