SEA-PHAGES | A. globiformis Digest Issues?

Link to this post \| posted 17 Oct, 2024 13:31
DrCatalase	Hello All, This is our first time using A.g. and we got good DNA extraction using the basic protocol from the discovery guide. However, when we tried digestion with NspI and SacII there were no DNA fragments just the large band of uncut DNA. Is there any trick or should I just try a longer digestion time? Thank you! Sean

Link to this post \| posted 17 Oct, 2024 13:56
viknesh	DrCatalase Hello All, This is our first time using A.g. and we got good DNA extraction using the basic protocol from the discovery guide. However, when we tried digestion with NspI and SacII there were no DNA fragments just the large band of uncut DNA. Is there any trick or should I just try a longer digestion time? Thank you! Sean Hi Sean, Are you seeing this for one phage or for multiple phages from your class? It's not uncommon for a 6-bp cutter like NspI and SacII to not cut phage DNA at all. In fact, this was true for several phages isolated by faculty using A. globiformis at the summer training this year. For that reason, we encourage using a 4-bp cutter. Typically, we recommend using HaeIII, which almost always cuts DNA, which reassures you that your reactions were setup correctly. Thought much less common, if phage DNA is modified, say at the C, then even HaeIII might not cut DNA. To help us decipher such interesting results, more recently, we've encouraged faculty to also include a second 4-bp cutter, MseI; HaeIII and MseI are GC and AT cutters respectively. I dont think longer incubation times are necessary. Instead, I'd recommend two things. First, include HaeIII (and MseI too, if you can). Second, maybe test your NspI and SacII on any DNA that you know they will cut (eg. NspI will cut pUC19 plasmid DNA).

Link to this post | posted 17 Oct, 2024 13:56

viknesh

DrCatalase
Hello All,
This is our first time using A.g. and we got good DNA extraction using the basic protocol from the discovery guide. However, when we tried digestion with NspI and SacII there were no DNA fragments just the large band of uncut DNA. Is there any trick or should I just try a longer digestion time?
Thank you!
Sean

Hi Sean,

Are you seeing this for one phage or for multiple phages from your class?

It's not uncommon for a 6-bp cutter like NspI and SacII to not cut phage DNA at all. In fact, this was true for several phages isolated by faculty using A. globiformis at the summer training this year. For that reason, we encourage using a 4-bp cutter. Typically, we recommend using HaeIII, which almost always cuts DNA, which reassures you that your reactions were setup correctly. Thought much less common, if phage DNA is modified, say at the C, then even HaeIII might not cut DNA. To help us decipher such interesting results, more recently, we've encouraged faculty to also include a second 4-bp cutter, MseI; HaeIII and MseI are GC and AT cutters respectively.

I dont think longer incubation times are necessary. Instead, I'd recommend two things. First, include HaeIII (and MseI too, if you can). Second, maybe test your NspI and SacII on any DNA that you know they will cut (eg. NspI will cut pUC19 plasmid DNA).

Link to this post \| posted 17 Oct, 2024 13:58
DrCatalase	Perfect, thank you Vic!

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A. globiformis Digest Issues?