Problems of LentiCRISPR V2 digested with BsmBI

[sif]

 Hello everyone, please help!

 

I recently did a GeCKO experiment by using the lentiCRISPR V2 (addgene#52961), the first procedure was to insert the sgRNA into the BsmBI digested lentiCRISPR V2. According to the lentiCRISPR v2 Notes in addgene website https://www.addgene.org/52961/notes/, when digested with BsmBI, we can get two fragments, 1.8kb and 12.9kb, we can purified the 12.9kb fragment and used it for the next procedure. However, I can’t observed the two expected bands after digested lentiCRISPR v2 plasmid with FastDigest Esp3I (isoschizomer of BsmbI) enzyme from thermo fischer. I got five different fragments after digestion (about 5000bp, 3000bp, 2000bp, 800bp and 600bp, respectively), I am uploading gel picture where,

Lane 1, 1kb plus DNA ladder

Land 2, lentiCRISPR V2 digested with FastDigest Esp3I for 30min

Lane 3, lentiCRISPR V2 digested with FastDigest Esp3I for 15min

Lane 4, lentiCRISPR V2 digested with FastDigest Esp3I for 5min

Is there anybody experienced the same problem? What might be the reason for this result? 

Thank you tremendously for any advice.

Fusheng

2-1-2017

[ray.n...@gmail.com]

To be clear, have you tried the digestion with regular BsmBI? If not, you should include that as a control.

[sif]

hi, i have not tried the digestion with regular BsmBI, thank you for your suggestion, i will try it.

在 2017年2月2日星期四 UTC+8下午11:37:58，ray.n...@gmail.com写道：

[Nektaria Μ. Leli]

Hello, I am doing exactly the same thing as you did and have no ptoblems. I think the plasmid you are cutting is not the GeCKO V2.