List the biological process(es) addressed in this protocol. List the methods used to carry out this protocol (i.e., for each step). If the gene probe is longer than 400 nt, increase the limiting nucleotide (UTP in this case) concentration to 5–25 μM by adding more non-labeled UTP in the transcription reaction. Wrap the membrane with saran wrap and expose to a phosphor screen overnight. Take out the membrane and brief dry it on kimwipe. Wash with high stringency for 15min twice. Pre-warm both the low stringency and high stringency buffer at 68 ☌. Spin at 700g for 2 min.Īdd 1 μl of G-50 column purified probe to 5 ml scintillation liquid and check the counts of the probes.įor gene probe, add 10 6 cpm/ml to fresh hybridization buffer for marker probe, add 0.2 × 10 6 cpm/ml.ĭump the pre-hybridization solution and add hybridization buffer with probes to the hybridization bottle. Load transcription reaction carefully on top of the G-50 column. Snap off the bottom of micro G-50 sephadex spin column open the cap by ¼ circle and put the column in a 1.75 ml tube. For a 20 μl gene probe transcription reaction, combine 10–50 pmoles DNA template, 2 μl 10× transcription buffer, 1 μl 10mM ATP, 1 μl 10 mM GTP, 1 μl 10 mM CTP, 5 μl 12.5 mM -UTP and T7 polymerase (20 U/μl final) in a PCR tube.įor a 20 μl transcription reaction for Millennium Marker probe, combine 1 μg DNA template, 2 μl 10× transcription buffer, 1 μl 10mM ATP, 1 μl 10 mM GTP, 1 μl 10 mM CTP, 1 μl 10 mM UTP, 2 μl 12.5 mM -UTP and T7 polymerase (20 U/μl final) in a PCR tube.Īdd 1 μl DNase I and incubate at 37 ☌ for 15 min to digest the template. Incubate at 68 ☌ for 1hr to pre-hybridize.ĭuring pre-hybridization, start the probe transcription reaction. Put cross-linked nylon membrane in the hybridization bottle with the RNA-side up.Īdd 10 ml hybridization buffer (for small hybridization bottle) to the membrane. Nonspecifically bound probes are washed away after hybridization. The probes are then hybridized to the membrane. Use in vitro T7 transcription to make radioactively labeled RNA probes complementary to RNA transcript of interest. Gel can be illuminated with UV to check whether there is any remaining RNA. UV crosslink the membrane twice to fix the RNA on the membrane and use a fine marker to mark the edge of the side with RNA.Ĭlean the gel transfer system thoroughly by rinsing with plenty of H 2O.Īfter the gel transfer, the gel area inside the window of green gasket should be half as thick as the gel outside the window. Dry the nylon membrane between two sheets of filter paper. Remove the gel and take out the nylon membrane. When the transfer is over, remove the sealing frame and drain the buffer. Occasionally check the buffer level to make sure it is above the gel. Place the lid on and transfer for 90 mins at 5 inches of Hg. Gently pour 1 L of 10× SSC buffer into the reservoir. Press the gel and along the window gently to apply extra pressure to help the vacuum sealing. Start the vacuum source and adjust the pressure to 5 inches of Hg. Place the sealing frame on top of the vacuum stage and lock it. Remove all the air bubbles between gel and the nylon membrane. Also make sure the gel overlaps with the gasket by at least 5 mm. Gently place the gel on top of the gasket with the well-side up. Make sure the gasket covers the seal o-ring while the membrane/filter paper overlaps with the window of the gasket. Place the plastic gasket on top of the membrane/filter paper. Wet the seal o-ring on the base unit with H 2O. Make sure there is no air bubble between membrane and filter. Place the wetted nylon membrane on top of the filter paper. Put wet filter paper on the vacuum porous stage and make sure the filter paper is in the area where the cut window of the green plastic gasket is going to be. Wet the nylon membrane and filter paper first in H 2O and then 10× SSC buffer Cut a filter paper with the same size as the nylon membrane.įill the wells of the RNA gel with melted agarose. RNA is transferred from gel to nylon membrane using vacuum gel transfer system.Ĭut a nylon membrane about (or bigger than) the size of the denaturing RNA gel.
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