Dear professional experimental masters,
As you can see from the schematic diagram (front view of a gel, white part is the DNA which showed on blue light after staining), there is a problem of the depth of bands after running a gel. For example, I added the sample into the pore of gel, it was 5mm depth. But after running a gel, and cut the band, it is only
Dear Jun, It is always difficult to run the perfect gel when you need to - especially when you want a good photograph - but if you are able to isolate the band of interest it may not matter so much how good the gel looked before you cut it out. Your problem may simply be that your sample is not sufficiently dense to sink all the way to the bottom of the well before you start the electrophoresis. You could 'pre-try' a gel by adding some normal loading buffer (i.e. 2 ul of a 10x buffer plus 18 ul water) and waiting to see if it sinks to the bottom within a minute or so. If it doesn't, you could try to gently wash the well out with electrophoresis buffer and try again (if it does work, then wash out the dye sample with electrophoresis buffer before loading your real sample). I suspect that the problem may be in how quickly you remove the comb after pouring the gel - if the agarose is not completely set, some will remain in the well after you have removed the comb and this will prevent your sample from sinking to the bottom! Regards, Andrew.
The migration of the DNA through the gel is not going to affect the gel extraction efficiency. You are probably either: not loading enough DNA into your wells, or not properly dissolving the agarose slice that contains your desired DNA. You need to cut carefully to minimize the amount of agarose also.
Good luck!
Doctor Spiers, thank you very much! The advices will be very helpful!
The density might be a problem. I used 50ml 2% agarose, and every pore was input by 30µl well mixture (25µl sample plus 5µl 6X loadding buffer). As soon as inputing, I turn on the power and started to run the gel in a minute. I will check it following your method.
The comb was removed about an hour after pouring the gel. Maybe it will not be a problem.
Thank you again. ^_^ Andrew J Spiers
Doc. Burnette, thank you very much for your advices! Katie A Burnette
The concentration of template DNA samples in this test experiment (18S V4 region), are only 5 to 9 ng/µl. I tried to use AMPure beads clean-up as contrast, >70% recovery rate as a result. However, nearly nothing was gotten after gel extraction.
The gel was dissolved well and cut as the smallest part which contained with DNA.
Doctor Spiers, I have tried the experiment following your advices, result is in the first figure. It seems that there is no problem with the density.
So I ran 3 ladder (5µl, 20µl and 20µl+4µl loading buffer) on 50ml 2% agarose gel for 30min (100V). And cut the band for every length of DNA in the gel, to see if the depth change in different length of DNA. Result is in the second figure. And it is very strange that there is no band showed with 5µl ladder.
Maybe the DNA seperate from the loading buffer when running?
Hi, everyone who has followed this question. I have found the main problem...It is actually there are not enough ethanol in the washing buffer of the gel extraction kit. Finally the product from the gel could be detected, but the recovery rate is a little low (20%-40%). But at least there are something after gel extraction!
Thank you for all of the advises!
Excellent problem solving Jun Xia ! Glad you are able to get the PCR product recovered from your gel.
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