Hosuk:LabNotes/2013-4-1
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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.