I am digesting pCS2+MT plasmid with Xhol-Xba1 double digestion. Previously it was ok but now the gel image like dot and smearing ? So ligation failure ! Could you please tell me the reason? I am attaching detail in the question doc below...
Change the water and buffer, its due to contamination. Apart from that confirm the quality of plasmid on gel before digestion.
Hi there,
It seems there is a lot of material on the gel... How much plasmid do you load onto the gel?
Hi, I agree with Dominique. A large amount of material have problems during the running and can caused the dot (or bubble) and some smear, but I don't see a classical smearing caused by degraded material; you have, perphaps, some undigested plasmid.
So if you have to digest and run this amount of plasmid, I think you can solve the problem loading in a more large well, but if you don't have the appropriate comb you can unite two wells with an adhesive tape ( I used this method several time and it's ok!) or loading in two wells. I also want to say that maybe you can digest less material or at least try also an ON digestion, I use 2h of digestion for only 2-3ug. I don't know the detail of your ligation failure, but a lot of time is undigested plasmid fault.
Tank you everyone.
Plasmid about 5ug. I also think there are lot of materials. After RE digestion and phenol-chloroform and ethanol precipitation (
1. Add 10 percent of 3M Na- acetate solution and 75-100 ul Phenol chloroform 50/50 solution to 100 ul sample and vortex well.
2. Centrifuge at 14000 rpm for 10 minutes.
3. Take upper layer about 100 ul gently.
4. Add 2.5 volumes of 100 percent ethanol and vortex well.
5. Keep in -800c for 15 minutes
6. Centrifuge at 14000 rpm for 15 minutes and discard or aspirate the supernatant.
7. Add 200ul 70 percent ethanol vortex and keep in -800c for 5 minutes
8. Centrifuge at 1400 rpm for 5 minutes and discard or aspirate the supernatant.
9. Air Dry pellet for 3-5 minutes
10. Resuspend the pellet in Nuclease free water – 10 ul)
I found the white materials in step 7 not dissolving well in 70 percent ethanol.
Another thing I noticed that the DNA materials migration through the uppermost layer of the gel !!
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