Anyone met this T7E1 issue?

[Aaron Tang]

Hello guys,

I did a T7E1 assay to confirm the effciency of sgRNA-1, 2. I did genomic PCR and purify the target band (~651 bp) from WT and modified gDNA. After that, I measured the concentration of PCR products and have 250 ug DNA from WT and modified to form heterduplex. And I digested the DNA duplex with/without T7E1, and finally had this weird result. There were 2 bands in the undigested sample (one is the 651bp duplex band, but another ~300bp which I don't know where it comes from). After the T7E1 digestion, the heterduplex were not digested, but the 300bp band was disappeared. But I confirmed that these sgRNAs worked because I did a RFLP analysis of the purified gDNA, and the sgRNA can destroy the enzyme site near the cleavage site. So the purified gDNA worked well. Did anyone met this T7E1 digestion issue? Thanks!

Ps. my protocol for heterduplex formation is as this:

95ºC for 10 min, 95ºC to 85ºC (- 2.0ºC/s),

85ºC for 1 min, 85ºC to 75ºC (- 0.3ºC/s),

75ºC for 1 min, 75ºC to 65ºC (- 0.3ºC/s),

65ºC for 1 min, 65ºC to 55ºC (- 0.3ºC/s),

55ºC for 1 min, 55ºC to 45ºC (- 0.3ºC/s),

45ºC for 1 min, 45 ºC to 35ºC (- 0.3ºC/s),

35ºC for 1 min, 35ºC to 25ºC (- 0.3ºC/s),

25ºC for 1 min, hold at 4ºC.

      

[Peter Romanienko]

It may be ssDNA left over from the denature/annealing. T7 endo, I believe, will cut up ssDNA.

[Mike]

You could confirm or exclude this possibility by doing an additional control: PCR product purified but not denatured/annealed (heteroduplex formation protocol). In that lane you should have no 300bp band as all the PCR product will be dsDNA. If the 300 bp product only appears after this, I guess Peter would be right, but I've never seen this before myself. I've seen other weird and annoying things with T7EI before though, that's why I switched to TIDE: http://tide-calculator.nki.nl/ I highly recommend giving this a try, for the sake of 2 sanger reactions on the PCR products you already have.

[Aaron Tang]

Yes, I think it is the ssDNAs as Peter said. I tried again. This time I didnot recover the gPCR product from gel, instead I directly use the gPCR reaction for denature/anneal, and this time works. But I don't know why recovered DNA cannot be re-annealed. Maybe the EB binding to the dsDNA in the gel matters.

在 2016年8月11日星期四 UTC-5上午10:20:44，Mike写道：