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Clean up Labeled Probes
Prewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.
Add 450ml miliQ H2O to each of the probe samples (or total 500μl). Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters. (Amicon)
Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.
Spin 10 minutes at 12,000 RPM
Repeat step 4 , spin 12min to get smaller volume.
Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
Transfer recovered probe to the appropriate partner probe.
Note: Probes can be combined after the first wash step.
Probe can be stored at 4℃ or -2℃ in dark for further purpose.
Reagents and Suppliers
| Cy3-dUTP |
1mM |
Perkinelmer |
NEL578 |
| Cy5-dUTP |
1mM |
Perkinelmer |
NEL579 |
| SuperScript II |
200U/μl |
Invitrogen |
18064-014 |
| RNA sin |
20-40U/μl |
Promega |
N2515 |
| 100 mM dNTP set** |
10X |
Amersham |
27-2035-01 |
| pd(N)6 Sodium Salt (Hexamer) |
50U |
Amersham |
27-2166-01 |
| Microcon YM-30 column |
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Amicon |
42410 |
Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH, 1M Tris-HCl pH7.5, sterile dH2O and DEPC H2O
* comes lyophilized, must be resuspended at specified concentration.
**for 10X stock: 5 mM each of dA, dG, dC and 2 mM of dT in DEPC H2O
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