Hosuk:LabNotes/2013-4-22
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Next Plan[edit]
RCA test[edit]
- Run RCA reaction in a tube with CircLigase II reaction sample using custom DNA
- Since the custom DNA has the sequence for binding to RCA primer,
- I could see the signal from RCA product.
Experiment plan for RCA test[edit]
- Run CircLigase II with custom ssDNA (2hr. @ 60C)
- Run Exo I (1hr. @ 37C) (--> Gel running)
- Run RCA
- Tube1 : custom ssDNA + RCA (18hr. @ 30C)
- Tube2 : Circ + RCA (18hr. @ 30C)
- Tube3 : Circ and Exo I + RCA (18hr. @ 30C)
Jay's advice[edit]
- (As following Jay's suggestion, 18hour RCA was done from yesterday to today using fixed cells, but couldn't get any reasonable result. There were some FL signal, but it was false signal)
- Jay's suggestion
- for now, just wash the cells well before you fix. (2 ml PBS x 3)
- after fixing cells and treating with etoh, wash with PBS.
- then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min.
- wash with 2xSSC several times.
- then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies.
- Jay's suggestion
CircLigase II test to prove what is circularized DNA[edit]
Sample set[edit]
- Run CircLigase II reaction
- input ssDNA : Custom ssDNA (50bp) having adapter sequence
- +CircLigaseII and -CircLigase II
- Reaction condition : 60C Thermo Cycler for 1hr 30min. --> 80C for 10min
Reaction condition[edit]
- CircLigase II : 20uL total reaction volume
Reagent | Conc. | Volume | Final Conc. | |||
ss DNA | 2uM | 5uL | 0.5uM | |||
10x Reaction Buffer | 10x | 2uL | 1x | |||
MnCl2 | 50mM | 1uL | 2.5mM | |||
Betain | 5M | 4uL | 1M | |||
CircLigase II | 100U/uL | 1uL | 5U/uL | |||
H2O | 7uL |
- Run Exo I reaction
- 9uL from CircLigase and CircLigase II reaction
- add 1uL of Exo I
- Run 37C for 1hr. --> 80C for 15min
Result[edit]
- TBU gel (All samples were denatured by 75C for 8min)
- Result
- -CircLigase II showed the same band to Custom ssDNA,
- and Exo I reaction of –CircLigase II showed nothing (Lane 7).
- Thus, the band in +CircLigase II and Exo I reaction of +CircLigase II are circularized DNA.
- In result, circularized 50bp of custom ssDNA (CircAdapt2 Primer) runs faster than linear one.
- This results are consistent with the previous results.
- RCA reaction is running with two samples, +CircLigase II and Exo I of +CircLigase II.
- Gel table
#1 | #3 | #4 | #5 | #6 | #7 | ||||||
10bp | Custom ssDNA | +CircLigase II | Exo I of +CircLigase II | -CircLigase II | Exo I of -CircLigase II |