I'm doing some cloning after longer pause and I'm having some troubles with my DNA agarose gels.
When doing colony PCR with QuickTaq HS DyeMix ( https://www.toyobo-global.com/seihin/xr/lifescience/products/pcr_018.html ), you can notice, that there are double bands. Actually, as I have now noticed, there's even smaller band, so it's probably not problem with the gel, but with the PCR?
Second, my DNA ladder looks weird. I'm still trying to find the right amount, it's either too diluted (not visible), or the bands are so thick that they are hardly resolved. I have further diluted it about 10x, so that I load 5 ul on the gel. Now, that I'm thinking about it, I have added third picture, where the bands next to the ladder are distorted (but curiously only the top bands), if that could be related? But those samples were done with other polymerase and mixed with dye by me.
Thanks for any insight in advance!
I think that your ladder is has too much salt ( possibly added with the loading dye) causing the smiling and that some of the smiling is also caused by running at too high a voltage. The ladder has run a long way into the gel. Try running at half the voltage . If there is too much salt in that ladder track then it is spreading during electrophoresis and pulling the closest dna sample into an unusual shape. The weak smaller band is probably primer misannealing and running the pcr at a higher annealing temperature or in the presence of dmso (less than 10% concentration) may get rid of this effect. It is hard to comment on the pentr image without knowing the sizes of the bands shown and the expected size of the amplimer but if the very strong band is your pcr amplimer then you may be running too many cycles of pcr. Is there a plasmid only control pcr sample in that picture to help sort out specific insert containing dna from plasmid only dna amplification?
Paul Rutland thanks for the extensive answer.
I have mixed the DNA ladder only with water and loading dye. Does the loading dye contain salt? What I have found, that it can contain 10 mM Tris/HCl and up to 100 mM EDTA, but problems with smears should be above 1M salt.
I run the gels at 100V (the distance between the wires is 10 cm, it's not the width of the gel, right?). Unfortunately, the systems we have only allow either 50 or 100V (or 150V). Would be 50V better?
On a side note, I have just found some table of conditions for agarose gel and there they state that for fragments
Loading dye. Most people make up their own loading dye and use 1/10th volume of loading dye added to the sample to be loaded so adding too much or errors in making up the dye ( like using 10xbuffer not 1x buffer) can cause salt problems. If you are using commercial loading dye and the right volume then do not worry about this.
Voltage. 50 volts will give better results but takes a bit longer.
You will probably never see a difference between TAE and TBE. Both are excellent.
Annealing. Ideally you should run a gradient of annealuing temperatures if you have a gradient pcr machine then use the cleanest amplification temperature but if you do not have a gradient machine then increase the temperature 2c at a time starting from 5c below the calculated annealing temperature of the lowest annealing primer. There are many online in silico pcr programmes that will calculate annealing temperatures but 50 is generally too low for clean amplifications.
If the sample next to the ladder is only plasmid then I cannot see any difference between the plasmid only amplification and the inserted plasmid. Perhaps you need primers one inside the insert and one on the plasmid to show that you have any insert within the plasmid. It looks like both of your primers are in the plasmid only sequence.
I will have a look at the other question Tomáš Hluska
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