Hosuk:LabNotes/2013-4-1

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Debugging rolony construction process[edit]

Cell fixation[edit]
  • 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[edit]
  • 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'
      • Order this primer, then hybridize after RT, and observe the signal.
  • 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[edit]
  • 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 a 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 test
    • 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 --> need to ask detail method how to hybridize circular template inside cells because there is not aminoallyl dUTP.
    • (4) Hybridize linear 50bp oligo in the fixed cells, circularization and RCA --> need to ask detail method how to hybridize 50 bp oligo inside cells because there is not aminoallyl dUTP.
    • (5) Compare rolonies (3), (4), and NTC (non CircLigase sample)


RCA[edit]
  • RCA reaction time
    • RCA is usually done for 20 hour reaction time, which would be enough. (I need to calculate if the amount of primers and dNTPs in the reaction mix are enough though)
    • Jay is doing exchange of fresh phi29 after 8 hour incubation, but he said this is not necessary.
  • Evaporation issue for long time incubation
    • There is no evaporation issue (20hr incubation), based on the observations after finishing RCA so far.
  • 30C incubation condition
    • Currently I've used Jeff's microscope chamber for 30C incubation.
    • Try to use 30C oven, if I need to do.
  • phi29 concentration
    • Jay said rolonies will be plenty even if low concentration of phi29 is used, thus this is not a problem
  • After doing those two validation - RT, and Circularization, I'll compare between RCA and NTC(without phi29), and different reaction time such as 1hr, 5hr, 10hr, 20hr.


Cell Fixation[edit]

  • 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.