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Figure 4 | BMC Molecular Biology

Figure 4

From: Fidelity of end joining in mammalian episomes and the impact of Metnase on joint processing

Figure 4

High fidelity repair in T-HO-CAT cells. (A) Episomes were recovered from cells after Ad-HO infection at 4 h and 6 h time point, subsequently used to transform bacteria, and plated on ampicillin plates. Boil prep of random clones from the 4 h and 6 h plates was performed to obtain the episomes and were run on 0.8% agarose gel to depict difference in size after repair (10.9 Kb in size). (B) SalI digestion of plasmids obtained from random clones of 4, 6, and 8 h plates PI was performed to reveal deletion in the range of 250–500 bp in size (Refer plasmid map and description in text). (C) PCR was performed across the DSB site using primers generating amplicon size of 120 bp with plasmids from random clones picked up from 4 h and 6 h time point plates and subsequently run in a 2.5% agarose gel to reveal micro deletions or insertions. (D) Accurately repaired clones were removed after isolating the episomes from cells at given time points PI by employing NheI digestion, thus enriching for inaccurately repaired plasmids (truncated ones). Episomes recovered from cells at indicated time points were digested with NheI overnight and subsequently transformed. Plasmids were subsequently obtained from random clones across different time points and were run on a 0.8% agarose gel. ‘U’ denotes uncut control plasmid. ‘C’ denotes SalI cleaved control plasmid.

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