Hi,
I am doing gene KO in Daphnia using CRISPR-Cas9 by microinjection. I did mutant screening using flanking primer in the DSB site producing 131 bp PCR product. I found one of my lines has two band that probably suggesting a heterozygote mutant. But I am curious about unexpected bands (red circle) that appeared with bigger size.
I am using phenol chloroform genome extraction for daphnia mutant, ExTaq Hotstart for the Genotyping PCR, and running the sample with Native PAGE.
Is there any idea regarding this matter?
Thee top 2 bands are more distant than I would expect but could they be heteroduplex bands of normal and mutant alleles?. You could cut out the strong smaller bands and mix them,denature at 94 and allow to re anneal and see if they produce the larger bands as well as their original size bands
Dear Paul,
Thank you for your valuable input. Yes indeed it still making the heteroduplex artefact after I did cloning. The heteroduplex will appear in colony PCR where both of the mutant and wildtype alelles are present. But, the clone than only has one of the original band will not produce the heteroduplex. It seem i can just ignore it. Thank you again for resolving my problem
Dear Ajay,
I did the sequencing but it seems it will not amplify the bigger band and only detect my lower band. So it probably true that the bigger band in question is artifacts.
thank you for the feedback Yusrifar K Tirta it helps me to learn also. The larger bands will not sequence well as after a certain point you have 2 sequences within each band so you will get 2 overlying sequences but at least the existence of the heteroduplex is useful if you ever need confirmation that a sample is a heterozygote
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