I currently have a problem with digestion reaction BmrI which I want to use to confirm my gene-editing experiment. My control with non-targeting should be 100% digested with BmrI while edited samples will be not. But there is a blur upper band in my non-targeting that makes my result not reliable.
My reaction performed 250ng DNA with 0.5ul of BmrI and 2.5ul NEBuffer™ 2.1 as recommended on the website. my reaction volume is 25ul, incubate 1h at 37oC and inactivate 65oC for 20 min.
I have tried to increase the restriction enzyme volume to 1ul, but no better.
I checked with sequencing that no mutation occurs to interrupt restriction.
I am looking forwards your advises and sugestion.
Thanks :))
Dear,
There is no strict rule, and the opinions might be varied. But below you can try!
1) Mix properly by applying gentle tapping at the bottom. Before adding the enzyme to the reaction, mix it properly and then add. Apply the second or third tapping in between the total reaction time.
2) Increase the incubation time.
3) If still, the problem exists, increase the reaction volume.
4) Check the purity of the DNA, run control or uncut one. Instead of TE buffer apply miliQ to collect the DNA and perform the digestions.
5) Take extra care during DNA isolation, so that no residual alcohol or protein contaminants exist.
6) Inactivate the restriction enzyme(e.g. applying heat) before running the gel.
Check http://www.hoajonline.com/journals/pdf/2050-2389-5-2.pdf
You need to compare your digestion to the expected DNA banding pattern. If the bands in both lanes are similar to the expected pattern and the additional bands are limited to spaces within the upper and lower bands of the expected pattern, the digestion is incomplete (partial) https://international.neb.com/faqs/2016/04/07/why-do-i-see-additional-dna-bands-on-my-gel-after-a-restriction-digest
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