Since you eluted too early in the protocol, your sample would have contained guanidine salts, agarose, and perhaps EDTA. When you dried it down, you also concentrated those chemicals. So you did get rid of the ethanol, but probably not the guanidine. If the contaminants were simply salts or EDTA, then ethanol precipitation would be a better way to clean up the DNA instead of drying it down.
If the contaminant was gel residue, ammonium acetate should be used for the precipitation salt because it will keep oligosaccharides soluble. If all of that chemistry is sounding like a bit too much hard work, you will be relieved to know that there is another solution that you could try if this happens again:. You can get more tips on gel extraction 15 in total!
But now that I am in gel extraction troubleshooting mode… here are a few more:. Always perform the additional QG wash to remove residual gel debris, especially if you pushed the limit of the max allowed for gel size, and then perform a second ethanol PE wash to make sure all the salt is removed.
Of course, dry the column before elution, but at least if you do forget, then the speed vac method will work to clean it up. Denatured alcohol contains chemicals such as isopropanol or benzene that do not evaporate quickly and this carries over into the DNA. To help dissolve gel, mix by vortexing the tube every 2—3 min during the incubation.
After the gel slice has dissolved completely, Add 1 gel volume of isopropanol to the sample and mix. Place a spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the column and centrifuge for 30—60 s. Discard flow-through. Place the column back into the same tube. For gel extraction, recommended: Add 0. This step will remove all traces of agarose.
It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection. To wash, add 0. Discard flow-through and place the column back in the same tube.
Centrifuge the column for an additional 1 min. A second problem centres on the size selective nature of this procedure. This is useful when signifcant amounts of primer-dimer and secondary amplifcation products are present. Alternative approaches:. In some cases where the PCR product is too small or alternative products interfere with successful sequencing, the isolation of the Target PCR product by gel purifcation is necessary. Complete removal of the media with a sterile pasteur pipette linked to the vacuum pump: after the first removal of the media lean the petri-dishes nearly vertically against a support for a short time so that the rest of the media can be collected from the bottom in the laminar flow.
Add 1 ml of TriZol Reagent Gibco: to the petri-dish and cover quickly the whole area by shaking - use sterile tip if possible with a filter included. This step should be done in the fume cupboard because of the phenol of the reagent.
If you want to prevent any potential contamination with RNases, you can do it in the laminar flow, too. Extract the cells by repeated pipetting of the solution over the whole area the solution usually gets a little bit turbid and transfer the extract to a sterile eppendorf tube.
Preparation of total RNA according to the protocol provided by Gibco. Centrifuge the tubes to get a drop at the bottom of the tube. Let the membrane dry for 10 min and then wet it a little bit from below using 5x SSC buffer.
Shake a little bit to dissolve completely; weigh the amount of water that is lost due to evaporation and fill up to the original weight if necessary heat again to dissolve completely and check the weight again. The gel is poured into the gel-bed this should be completely horizontal - check with bubble of spirit level and the sample comb is put in the sample comb is usually stored in ethanol to prevent RNase contamination and dried in the laminar flow before use.
Polymerisation of the gel is allowed for 30 min and then the gel is transferred to the electrophoresis apparatus filled with 1 l of 1xMOPS - diluted from 5x MOPS with nuclease-free water. The surface of the gel should be covered. The sample comb is carefully removed. Electrophoresis The samples are mixed with loading buffer at the bottom of the tube after centrifugation by repeated pipetting.
Then they are carefully filled into the sample pockets of the gel. Electrophoresis is carried out at 20V over night or at V for 3 h RNA migrates towards the plus-pole. The bromophenolblue should migrate about 8 cm, before ending the run. The gel is checked on the UV-monitor and a picture is taken with the Polaroid camera put a ruler to the gel. Partly RNase-digested samples migrate faster. Capillary Blot The gel is marked on the right lower edge, removed from the electrophoresis container and submersed in 0.
This is important for the transfer of RNA larger than 2. Wash the gel with nuclease-free water. Shake the gel for 45 min in 20x SSC-buffer. A tray is filled with 5x SSC, and a glass plate is put on the tray. Two Whatman 3MM filters 11 cm x ca. Air bubbles between the plate and the filters are removed by rolling a sterile pipette on the filter. The gel is put on the wet filters with the upper side down transfer is more efficient in this way; besides, the orientation of the samples on the blot is then equal to the orientation on the gel.
Air bubbles between gel and filters have to be removed with the pipette!!!! Air bubbles are removed again.
If the pre-cut 10 cm x 13 cm Whatman filters are used, the gel and the Hybond membrane have to be cut to this size; in this case just 13 instead of 15 samples can be applied to the gel.
Parafilm is put exactly to the edges of the gel, in order to prevent contact between the wet filters below the gel with the filters above the gel. A stack of dry Whatman filters height: 5 - 8 cm is laid on top, followed by a glass plate and a g weight bottle. The capillary transfer from the gel to the membrane should be carried out for 18 - 24 h.
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