Hosuk:LabNotes/2013-4-1

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Revision as of 23:15, 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
  • Most suspicious step
  • Reaction time
    • In the manual, 1 hour will be enough, so 2 hour reaction time would not be a problem


  • Mineral oil cover to prevent evaporation
    • I've tried both mineral oil and wet towel for 2 hour, 60C incubation
    • Result (observation of rolonies after RCA) was same, thus mineral oil cover, washing step are not problem.


  • The amount of CircLigase enzyme
    • The volume of CircLigase II enzyme was 2uL in the very first protocol that Jay sent.
    • However, in the phone call at 02-07, Jay said 2uL is ok because of expensive cost of the enzyme.
    • Original protocol in the manual of CircLigase II is 100 U per 20uL reaction volume, which means 5uL for 100uL reaction that currently I've used.
    • I will try 5uL of CircLigase II enzyme and do RCA and observe rolonies if there is any difference.


  • Circularization
    • I'll use 50 bp sequence having the adapter sequence that I'm using (TCTCGGGAACGCTGAAGA) and circularization, RCA and observe how rolonies are seen.
    • (1) order 50 bp oligo
      • 5' CAA GCA GAA GAC GGC A[TC TCG GGA ACG CTG AAG A]CT CGG CAT TCC TGC TG 3' (--> 50bp, sequence in the [] is the adapter sequence )
    • (2) Circularization test in the test tube using CircLigase II enzyme, gel running for verification
    • (3) Inject circular templates that are made from tube in the fixed cells, the RCA
    • (4) Hybridize linear 50bp oligo in the fixed cells, circularization and RCA
    • (5) Compare rolonies (3), (4), and NTC (non CircLigase sample)


RCA


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.