Hosuk:LabNotes/2013-4-22: Difference between revisions
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====Next Plan==== | ====Next Plan==== | ||
=====RCA test===== | |||
*Run RCA reaction in a tube with CircLigase II reaction sample using '''custom DNA''' | *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, | *Since the custom DNA has the sequence for binding to RCA primer, | ||
Line 5: | Line 6: | ||
**(As | =====Experiment plan for RCA test===== | ||
**Run CircLigase II with custom ssDNA (2hr. @ 60C) | |||
**Jay's | **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) | |||
[[File:RCAReationsinTube.jpg|600px]] | |||
=====Jay's advice===== | |||
*(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) | ***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. | ***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. | ***then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min. | ||
***wash with 2xSSC several times. | ***wash with 2xSSC several times. | ||
***then just do your RCA. (with aminoallyl dUTP), followed by the | ***then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies. | ||
====CircLigase II test to prove what is circularized DNA==== | |||
=====Sample set===== | |||
*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===== | |||
*CircLigase II : 20uL total reaction volume | |||
{| {{table}} | |||
| 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===== | |||
*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 | |||
{| {{table}} | |||
| #1 | |||
|| | |||
| #3 | |||
|| | |||
| #4 | |||
|| | |||
| #5 | |||
|| | |||
| #6 | |||
|| | |||
| #7 | |||
|| | |||
|- | |||
| 10bp | |||
|| | |||
| Custom ssDNA | |||
|| | |||
| +CircLigase II | |||
|| | |||
| Exo I of +CircLigase II | |||
|| | |||
| -CircLigase II | |||
|| | |||
| Exo I of -CircLigase II | |||
|} | |||
*[[File:Gel_CircII_Adapt2_2b.jpg|350px]] |
Latest revision as of 00:22, 24 April 2013
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 |