I am analyzing samples transfected by CRISPR using the T7 assay. And when I am trying to analyze it on the gel, I can found smears on my samples. I don't know if I added too much enzyme or very long incubation time. Any suggestions?
Here is my recipe/protocol used.
purified PCR product -150ng
NEB buffer 2- 1uL
Water- 9.5uL
Reaction 95C-10min, 85C- 5min (0.1C/sec), 65C-2min (0.1C/sec), 45C-2min (0.1C/sec), 25C-hold
Add 0.5uL T7 enzyme. Incubate for 1hr. Stop reaction by adding 1.5uL 0.25M EDTA. Run on gel.
thank you.
Reaction:
Did you also run an untreated (T7 Endonuclease) DNA sample for comparison? If yes, were they fine (not degraded)?
Thanks Yuan-Yeu Yau.
Yes, I did check the PCR result first before purifying. And some of them has unspecific bands. But the Wild type has a very clear sharp band, so I don't know on how to optimize the PCR reaction to eliminate the unspecific bands. My thoughts are the unspecific bands affect the T7 causing for the smears. By the way, I am using HIFI Phusion polymerase for my PCR with an annealing of 62C. Should you think I increase more the annealing temp or shorter the no. of cycles (30 cycles)? My doubts is that I may get lower concentration of PCR product.
Hi Christina,
1. I am not sure that the unspecific bands will affect the T7 and cause the smears. T7 endonuclease I only cut the unmatched bases of a DNA molecule (see attached figure).
2. Since your DNA sample contains unspecific bands, you should gel-extract and purify the right band first, then use it for T7 treatment. Please see this useful website ( http://www.crisprflydesign.org/t7-endo-i-assay/ ) for step-by-step protocol of T7 assay. They also suggest cut out the right band.
3. They used 10U of T7 endonuclease I, how much U did you use?
4. If you want to search a way to produce a clean single band, you can try to use a higher annealing temperature, but this depends on what are the Tm of your 2 primers. You don't want to over-shoot it.
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