Hosuk:LabNotes/2013-4-1

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Revision as of 22:54, 1 April 2013 by >Hosuki78
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Debugging rolony construction process

Cell fixation
  • Are mRNA enough in fixed cells?
    • Based on the result of polyA FISH (done at 03/17), there are plenty of mRNA in the fixed cells
Reverse Transcription
  • mRNA degradation --> I don't think this step has any problem...
    • I've cleaned bench and glass pipette for aspiration with RNAase-free and 70% ethanol, and UV the PBS, 0.1N HCl before starting.
    • After 0.1N HCl incubation for 10min, cells were washed with PBS 3 times.
    • To verify RT works well or not, Cy5 labeled primer will be used to visualize cDNAs
      • Primer for RT --> 5' 5Phos - TCT CGG GAA CGC TGA AGA NNN NNN 3'
      • Labeled primer --> 5' Cy5 - TCT TCA GCG TTC CCT AGA 3'


  • Reaction time was 8 hour
    • Based on the polyA FISH protocol (1 hour hybridizatoin time) over 1 hour incubation time could ensure fully hybridization of primers to mRNA.
    • 2000 units RTase in 200uL reaction mix are used, and 50 nmole dNTPs are used,--> 8 hr incubation time was very long time for cDNA extention.
    • Unit definition(from Enzymatrics --> 1 unit is defined as the amount of enzyme required to incorporate 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using poly r(A)/oligo (dT) as a substrate.
    • To find better RT reaction time, try different reaction time per each sample and use Cy5 labeled primer to see which sample has more cDNAs
      • (First of all I need to verify that the way using Cy5 labeled primer works)


Circularization

Cell Fixation

  • Two dishes were fixed.
  • The cells were split by Alan at 03-26,and they have been cultured until today, media was exchanged twice.
  • Cells are now more denser and plenty.