Hosuk:LabNotes/2013-4-23
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RCA test with the product of CircLigase II reaction[edit]
Goal[edit]
- Verify that there are circularized DNA in the CircLigase II reaction sample
Experiment Procedure[edit]
- Run RCA
- S1 : CircLigase II reaction sample (18hr. @ 30°C)
- S2 : -CircLigase II sample (No CircLigase II Enzyme while CircLigase II reaction) (18hr. @ 30°C)
- Protocol
- File:RCAReationProtocolinTube.jpg
- Mixing Cy3-Adapter
- 4.5uL of each RCA sample + 0.5uL of 100uM Cy3-Adapter
- S1 : +CircLigase II + Cy3-Adapter
- S2 : -CircLigase II + Cy3-Adapter
- S3 : H2O + Cy3-Adapter
- small drop(0.6uL) of each sample on a glass slide and cover with cover slide, and imaging
Result[edit]
- According to the image of RCA with +CircLigase II, there are RCA product.
- So, CircLigase II reaction was done.
- Again, circularized 50bp DNA ran faster than linear in the 20% TBU gel.
S1:RCA, +CircLigase | S2:RCA, -CircLigase | S3:H2O | |||
(Original) | (Original) | (Original) | |||
File:CircII Custom RCA Cy3Adapter 20x fig02.jpg | File:-CircII(noenzyme) Custom RCA Cy3Adapter 20x fig02.jpg | File:H2O Cy3Adapter 20x fig02.jpg |
Next Plan[edit]
- Test in-situ RCA using circularized DNA into fixed cell
- Add circularized DNA into fixed cell, and incubte for 15min at RT
- Wash 2xSSC several times (maybe twice)
- Run RCA (including aminoallyl dUTP)
- Priming Cy3-Adapter and image the Rolonies