SEA-PHAGES | All posts created by Lafayette

Link to this post \| posted 08 Oct, 2021 16:14
Lafayette_SEA-PHAGE	Lafayette has an opening for a Teaching and Research Support Specialist position to begin in January 2022. We are particularly interested in candidates with SEA-PHAGES experience. Check out the link below and please share this info with those who might be interested. Contact Jim Dearworth (dearworj@lafayette.edu) if you have questions. 16Kb

Posted in: General Message Board → Staff position at Lafayette College

Link to this post \| posted 15 Nov, 2016 21:36
Lafayette_SEA-PHAGE	Viknesh Sivanathan Hi Laurie, Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C. Vic Today we started with a brand new kit and new isopropanol, and got even lower concentrations of DNA (and the 260/280 was also low). We are now back at the point of making new webbed plates to get more phage for another DNA isolation. We will try the PEG precipitation with these. We were just hoping that a new kit and new isopropanol would increase our concentrations. Laurie

Link to this post | posted 15 Nov, 2016 21:36

Lafayette_SEA-PHAGE

Viknesh Sivanathan
Hi Laurie,

Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C.

Vic

Today we started with a brand new kit and new isopropanol, and got even lower concentrations of DNA (and the 260/280 was also low). We are now back at the point of making new webbed plates to get more phage for another DNA isolation. We will try the PEG precipitation with these. We were just hoping that a new kit and new isopropanol would increase our concentrations. Laurie

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 11 Nov, 2016 16:56
Lafayette_SEA-PHAGE	Viknesh Sivanathan Hi Laurie, Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C. Vic Only three students have gotten to the DNA isolation stage. A few more will get there by Tuesday (I hope). I'll try the PEG precipitation; hopefully that will do the trick. Laurie Edited 11 Nov, 2016 16:56

Link to this post | posted 11 Nov, 2016 16:56

Lafayette_SEA-PHAGE

Viknesh Sivanathan
Hi Laurie,

Is this true across the board, or are only 3 students getting low DNA yields? A little more cumbersome, but you could try to concentrate the phage, either using the EM protocol for concentrating phage, or the PEG precipitation protocol. When concentrating, you should spin at 4C.

Vic

Only three students have gotten to the DNA isolation stage. A few more will get there by Tuesday (I hope). I'll try the PEG precipitation; hopefully that will do the trick.
Laurie

Edited 11 Nov, 2016 16:56

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 11 Nov, 2016 13:09
Lafayette_SEA-PHAGE	SBS_UNL My query is somewhat related to DNA extraction/recovery. Using the Wizard kit (kit is newly purchased; and we are following carefully protocol 9.1) we are getting meager yields of DNA for our lysates (titers = or > 10^9 pfu/mL; DNA averaging 20 to 25 ng/µL). At first I thought our lysates were 'old', but even extracting immediately after collecting lysates (a spot plate titer indicated a high titer for the fresh lysate) did not boost the yield. I will concentrate the recent lysates, and have the students extract the concentrate. Are we overlooking something? Any suggestions to increase the recovery of DNA? Cathy Chia at Univ Nebraska-Lincoln (Cohort 9) Viknesh Sivanathan Hi Rodney, I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in. In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes? Vic I'm also having very low yields, even lower than Cathy's. Three students are at DNA isolation stage and their concentrations are averaging about 10 ng/µl, two students have repeated the isolation with no change in concentration. We have been using a Wizard kit from 2015; I have a new kit that we can try. The only adjustment we tried the second time was to warm the resin to 37°C before use (this was from last years protocol), but that didn't help. We are struggling to get enough DNA for the electrophoresis and to ship next week. I'm hoping someone has some suggestions. Laurie Caslake at Lafayette College Edited 11 Nov, 2016 13:14

Link to this post | posted 11 Nov, 2016 13:09

Lafayette_SEA-PHAGE

SBS_UNL
My query is somewhat related to DNA extraction/recovery. Using the Wizard kit (kit is newly purchased; and we are following carefully protocol 9.1) we are getting meager yields of DNA for our lysates (titers = or > 10^9 pfu/mL; DNA averaging 20 to 25 ng/µL). At first I thought our lysates were 'old', but even extracting immediately after collecting lysates (a spot plate titer indicated a high titer for the fresh lysate) did not boost the yield. I will concentrate the recent lysates, and have the students extract the concentrate. Are we overlooking something? Any suggestions to increase the recovery of DNA?
Cathy Chia at Univ Nebraska-Lincoln (Cohort 9)

Viknesh Sivanathan
Hi Rodney,

I can't say I've seen this, and have not yet heard from anyone with this problem. Perhaps someone who has will chime in.
In the meanwhile, would you share a gel where you see the degradation only in the presence of restriction enzyme buffer? I assume the buffer isn't contaminated with DNAse, and that you are using brand new buffer when you used the new enzymes?

Vic

I'm also having very low yields, even lower than Cathy's. Three students are at DNA isolation stage and their concentrations are averaging about 10 ng/µl, two students have repeated the isolation with no change in concentration. We have been using a Wizard kit from 2015; I have a new kit that we can try. The only adjustment we tried the second time was to warm the resin to 37°C before use (this was from last years protocol), but that didn't help. We are struggling to get enough DNA for the electrophoresis and to ship next week. I'm hoping someone has some suggestions.
Laurie Caslake at Lafayette College

Edited 11 Nov, 2016 13:14

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 27 Oct, 2016 17:49
Lafayette_SEA-PHAGE	Hi, The EM at our buddy school, Lehigh University, is down and they are uncertain as to when it will be available. Does anyone have a recommendation and contact info for another school who might be able to photograph our phage? Laurie Caslake, Lafayette College

Posted in: Phage Discovery/Isolation → Electron Microscope access

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