Fragment analysis with Bioanalyzer or Fragment analyzer
This service enables you to check quality of your DNA or RNA (total, small or mRNA). The FGL employees both the Agilent 2100 Bioanalyzer and the Fragment Analyzer to visualize Nucleic Acids for quality control before and after library preparation. The FGL also houses a Femto Pulse for femtogram scale visulization of High Molecule Weight gDNA or low input mRNA.
Submitting samples for Bioanalyzer: Bring your completed form with your sample tubes to 255 LSA (We encourage to use a low binding tube). We receive samples until 6 PM. (If it is after 5 and you have no access to building, call the lab from outside and we can let you in.) Some things to keep in mind: Aim high on concentration, as many methods estimate concentration higher than the Bioanalyzer does, as the dye binds only to either single or double stranded molecules. Also signal quality is better with higher concentration. Try to use low-binding tubes. Also don’t have samples diluted in a surfactant solution. Results are available in 1-3 days
The Qubit® Quantitation Platform uses specific fluorescent dyes to detect the specific molecules of interest, so it is more accurate since it detects only the molecule of interest. However, the UV spectrophotometers (include NanoDrop®) use UV absorbance, which cannot distinguish between DNA, RNA, degraded nucleic acids, free nucleotides, and other contaminants. The UV absorbance can be unreliable and inaccurate especially at low concentration (<100 ng), and can be of DNA, RNA, protein, degraded nucleic acids, and free nucleotides. The Qubit® Fluorometer generates more accurate and precise results across a lower concentration range than those obtained by UV absorbance measurements on the NanoDrop® spectrophotometer. Accordingly, fluorescent quantitation of nucleic acids is recommended in the MIQE (Minimal Information for Publication of Quantitative Real-Time PCR Experiments) Guidelines.
Fast DNA Electrophoresis (H3BO3 or B2O3·3H2O) Using SB Buffer (Biotechniques36, 214-216, Feb, 2004)
- 20 X SB buffer(1L)-dissolve 8.0g of NaOH & 47.0 g of Boric acid in 1000mL (pH ~8.0), and filter with 0.2um bottle-top disposble filter. please make sure they are all disolved before filtering.
- make 1XSB buffer by diluting 20X solution.
- Use this SB uffer in the same way as TBE/TAE to prepare the agarose gel, and as the medium to run the gel in. Gels can typically be run at 300 V (around 10 min for a 10 cm gel). A 3% gel can be used to detect and separate short DNA (down to 40 bp).
- The gels do not need to be submerged since the SB has much higher conductivity. Pour the SB buffer into the gel tanks (& wells) to the gen level & run the gel.
– Brody, J. R., et al. (2004). Biotechniques 37, 598-602 / Brody, J. R., and Kern, S. E. (2004). Biotechniques 36, 214-216 / Goedhart, J. and Gadella, T.W., Jr. (2005). Anal Biochem, 343, 186-187.
Analyzing RNA on an Agarose Gel Under RNase Free Environment
(I learned this protocol when I was doing my postdo cat Rao’s lab at UC Riverside. I think you will find this is a useful protocol when you cannot access a bioanlyzer or when your RNA is concentrated enough to be detected by a dye (EtBr or Sybr green in gel).
- Make RNase free solutions for Agarose gel electrophoresis.
- RNase-kill solution (0.05%SDS & 0.05%)
- RNase free TBE or TAE by filtering with a 0.2µm bottle-top disposable filter.
- Put a comb, tray in the agarose gel tank
- Warm the RNase-kill solution by microwaving & cool to <65oC.
- Pour in to the agarose gel tank (step 2)
- Cover the gel tank with a sheet of a plastic wrap.
- Incubate 3hr~o/n.
- Warm RNase free water (Milli-Q water with filter installed or autoclaved) to ~65oC.
- Pour out the RNase-kill. Since the RNase-kill can be used many times, collect and filter.
- Pour the warm water to the empty tank, which contains all the necessary parts (a comb, tray, and barrier).
- Incubate at room temp & pour out the water.
- Repeat step 7~9.
- Set the gel on a sheet of a plastic wrap or in the tank.