SEA-PHAGES | Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 03 Nov, 2016 14:54
rodney_king	Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNAse is not being thoroughly removed.

Link to this post \| posted 03 Nov, 2016 18:35
viknesh	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 Edited 03 Nov, 2016 18:36

Link to this post | posted 03 Nov, 2016 18:35

viknesh

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

Edited 03 Nov, 2016 18:36

Link to this post \| posted 07 Nov, 2016 15:00
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

Link to this post | posted 07 Nov, 2016 15:00

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

Link to this post \| posted 07 Nov, 2016 15:37
rodney_king	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 Hi Vic, I am happy to share gel images. I will post them soon. Yes, we have used brand new buffers and enzymes. We also tested the "old" enzyme and buffers on DNA isolated using the old protocol and on plasmid DNA using a different DNA isolation kit. These DNA samples cut normally and there was no sign of degradation. Rodney

Link to this post | posted 07 Nov, 2016 15:37

rodney_king

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

Hi Vic,
I am happy to share gel images. I will post them soon. Yes, we have used brand new buffers and enzymes. We also tested the "old" enzyme and buffers on DNA isolated using the old protocol and on plasmid DNA using a different DNA isolation kit. These DNA samples cut normally and there was no sign of degradation.
Rodney

Link to this post \| posted 08 Nov, 2016 18:38
viknesh	Hi Cathy, Are you getting low yields across the board or just for a number of students/samples? Typically, one can expect a yield of ~ 40ng/ul or higher from a 10^9 pfu/ml lysate when prepping using the protocol 9.1. If a lysate yields low DNA, there is always the option to concentrate the lysate by centrifugation (see PEG precipitation, or the TEM protocol). Regardless of the protocol you use for concentrating phage by centrifugation, it will be important that you spin at 4C. If you are seeing low yields across the board, perhaps its because of a bad reagent. Vic

Link to this post | posted 08 Nov, 2016 18:38

viknesh

Hi Cathy,

Are you getting low yields across the board or just for a number of students/samples? Typically, one can expect a yield of ~ 40ng/ul or higher from a 10^9 pfu/ml lysate when prepping using the protocol 9.1. If a lysate yields low DNA, there is always the option to concentrate the lysate by centrifugation (see PEG precipitation, or the TEM protocol). Regardless of the protocol you use for concentrating phage by centrifugation, it will be important that you spin at 4C.

If you are seeing low yields across the board, perhaps its because of a bad reagent.

Vic

Link to this post \| posted 09 Nov, 2016 21:24
viknesh	Rodney King Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNase is not being thoroughly removed. Hi Rodney, I had a conversation with Steve Caruso today, and he's seen what you've been describing. He thinks it happens when students remove the resin/denaturant without first properly mixing the solution. As a result, the DNase added to the lysate/resin mix may not be fully denatured. Any remaining folded DNase may be inactive during purification because of the presence of EDTA. In the elution (in water), perhaps there isn't sufficient cations to support DNase activity. The addition of the restriction buffer, however, bring in cations that might support DNase activity. Thoughts? Vic Edited 10 Nov, 2016 00:28

Link to this post | posted 09 Nov, 2016 21:24

viknesh

Rodney King
Has anyone experienced degradation of DNA isolated by the new protocol? We tried a brand new DNA isolation kit and brand new enzymes and still see degradation. Never experienced this problem before. The fact that the DNA disappears only in the presence of restriction enzyme buffer indicates DNase is not being thoroughly removed.

Hi Rodney,

I had a conversation with Steve Caruso today, and he's seen what you've been describing. He thinks it happens when students remove the resin/denaturant without first properly mixing the solution. As a result, the DNase added to the lysate/resin mix may not be fully denatured. Any remaining folded DNase may be inactive during purification because of the presence of EDTA. In the elution (in water), perhaps there isn't sufficient cations to support DNase activity. The addition of the restriction buffer, however, bring in cations that might support DNase activity.

Thoughts?

Vic

Edited 10 Nov, 2016 00:28

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

Link to this post \| posted 11 Nov, 2016 15:08
viknesh	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

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

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

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Yield and degradation of DNA isolated by new protocol