SEA-PHAGES | DNA Smear

Link to this post \| posted 30 May, 2019 19:28
c.sunnen	Matt, Did you use our protocol? Your gel looks similar to ours. We also had some issues with sequencing - turns out we had some bacterial DNA contamination - but we assumed that was user error (asking the TAs to add the nuclease instead of doing it ourselves). We're actually going to test this over the summer. Otherwise, our DNA seemed pretty clean. Nikki

Link to this post \| posted 30 May, 2019 20:07
mastropm	Hi Nikki, Yes we used your protocol. I think our nuclease mix is not 100% correct. We are getting a lot of what is most likely RNA contamination. The one I posted had the most smearing and the DNA concentration was too low. The one that worked had the least smearing of all. 92Kb

Link to this post \| posted 30 May, 2019 20:07
mastropm	Hi Nikki, Yes we used your protocol. I think our nuclease mix is not 100% correct. We are getting a lot of what is most likely RNA contamination. The one I posted had the most smearing and the DNA concentration was too low. The one that worked had the least smearing of all. 92Kb

Link to this post \| posted 03 Jun, 2019 18:29
Thiel	Hello, This was my first time running the phage discovery semester and some of the DNA was smeared. We used M. foliorum as our host. We used the nuclease mix from Sigma-aldrich (#GE80-6501-421018-03) and did the EDTA, proteinase K, and used the wizard kit. We tried it the first time and ran uncut and cut with RE. All of the gels/ phages except one, were smeared. Renzie was the best and still smeared some. We isolated DNA again and just ran uncut. From the uncut DNA gel image, Dan was able to sequence DNA from Renzie and Lenlyn. Thanks, Sarah 926Kb

Link to this post | posted 03 Jun, 2019 18:29

Thiel

Hello,
This was my first time running the phage discovery semester and some of the DNA was smeared. We used M. foliorum as our host. We used the nuclease mix from Sigma-aldrich (#GE80-6501-421018-03) and did the EDTA, proteinase K, and used the wizard kit. We tried it the first time and ran uncut and cut with RE. All of the gels/ phages except one, were smeared. Renzie was the best and still smeared some. We isolated DNA again and just ran uncut. From the uncut DNA gel image, Dan was able to sequence DNA from Renzie and Lenlyn.

Thanks,
Sarah

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