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

Link to this post \| posted 16 Nov, 2016 15:12
DanRussell	Hi all, Since we at Pitt are receiving DNA samples for sequencing (and thus gel pictures as well) I can say that we're seeing more smeary degraded stuff this year than in the past. Not sure what's going on, but I don't think it's limited to 1-2 schools. –Dan

Link to this post \| posted 16 Nov, 2016 16:21
viknesh	Dan, here is a representative gel (of ~ 40 gels) from the workshop when faculty used the new DNA isolation protocol. As you can see, there is some amount of smear. Is this what you are receiving for sequencing? 140Kb

Link to this post \| posted 17 Nov, 2016 15:21
edgingtonn1	My students are having a very tough time getting a high enough yields with this new protocol! We may not make the deadline of tomorrow (11-18-16). We are following the protocol and keeping in mind all of the potential pitfalls with the kit. We have seen a little smearing on the "best" preps, and we have one prep that also does the disappearing trick after R.E. digestion. With the caveats of a NanoDrop in mind, we are getting ~40ng/uL to ~80ng/uL following the protocol, and in several cases when we did not split the one mL HTL (i.e. added 1mL of HTL to one column), the resulting yield was double (i.e. ~160ng/uL), which might be useful to know. Our lysate titers have been low as well, thus we have been working with lysates very close to 1x10^9 pfu/mL, because it has been the best that the students have been able to achieve this year (which is a separate issue to be sure.) Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Link to this post | posted 17 Nov, 2016 15:21

edgingtonn1

My students are having a very tough time getting a high enough yields with this new protocol! We may not make the deadline of tomorrow (11-18-16). We are following the protocol and keeping in mind all of the potential pitfalls with the kit. We have seen a little smearing on the "best" preps, and we have one prep that also does the disappearing trick after R.E. digestion. With the caveats of a NanoDrop in mind, we are getting ~40ng/uL to ~80ng/uL following the protocol, and in several cases when we did not split the one mL HTL (i.e. added 1mL of HTL to one column), the resulting yield was double (i.e. ~160ng/uL), which might be useful to know. Our lysate titers have been low as well, thus we have been working with lysates very close to 1x10^9 pfu/mL, because it has been the best that the students have been able to achieve this year (which is a separate issue to be sure.)

Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Link to this post \| posted 17 Nov, 2016 16:41
decourcy	Just found this thread, and thought I'd chime in. We too are seeing greatly reduced yields this year compared to last year. However, we are not having a problem with degradation.

Link to this post \| posted 17 Nov, 2016 17:00
DanRussell	Nicholas Edgington Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results. Hey Nick, We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows? Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help. –Dan

Link to this post | posted 17 Nov, 2016 17:00

DanRussell

Nicholas Edgington
Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Hey Nick,

We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows?

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

Link to this post \| posted 06 Dec, 2017 19:12
libby1954	All of our 8 students at Salish Kootenai College had degraded DNA when the uncut and enzyme digests were run on a gel. It appeared to be completely degraded, since there was only a small spot near the bottom of the gel. Only one student had some faint bands at the high weight at the size of uncut. I suspect that the DNAse was not inactivated, or that too much was added. One problem was when mixing up the DNAse (I did not use RNAse). The recipe card on page 86 of Phage Discovery Instructor's Guide, 2017 Edition, was unclear. In step 4 of "To Prepare" the final volume is brought to 5 mL. But the addition of the volumes of reagents listed (including water) equals about 9.3 mL. I have not seen any posts this year about problems with degraded DNA, but wonder if there are any recommendations. We have not tried the DNA isolation again yet, and the quarter is ending this week, so we will try again in January. Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use. Thank you, Libby

Link to this post | posted 06 Dec, 2017 19:12

libby1954

All of our 8 students at Salish Kootenai College had degraded DNA when the uncut and enzyme digests were run on a gel. It appeared to be completely degraded, since there was only a small spot near the bottom of the gel. Only one student had some faint bands at the high weight at the size of uncut.

I suspect that the DNAse was not inactivated, or that too much was added. One problem was when mixing up the DNAse (I did not use RNAse). The recipe card on page 86 of Phage Discovery Instructor's Guide, 2017 Edition, was unclear. In step 4 of "To Prepare" the final volume is brought to 5 mL. But the addition of the volumes of reagents listed (including water) equals about 9.3 mL.

I have not seen any posts this year about problems with degraded DNA, but wonder if there are any recommendations. We have not tried the DNA isolation again yet, and the quarter is ending this week, so we will try again in January.

Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use.

Thank you,
Libby

Link to this post \| posted 09 Dec, 2017 00:05
amandabbraley	Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help. –Dan Funny you should ask this, Dan. This is my winter break project! We had awful DNA degradation this semester! I was going to pull out old stocks of our nucleases and our old protocol and give it a try against the "new" nuclease mix and protocol. Looking at the shockingly high proportion of smeared restriction digests this week (as we're doing presentations), it suddenly occurred to me that maybe (just maybe) it was something about the MP Biomedicals™ Deoxyribonuclease I (from Fisher ICN10057520). We previously used Thermo Scientific™ Pierce™ DNase I (#89836)–as well as an EDTA step. We also had problems with low titers and our microscopist at WSU mentioned how "bad" our samples looked compared to years of other samples we've sent her. We're wondering if our M. foliorum phages aren't very stable in phage buffer or if the PYCa media system is causing some chemical reactions that are changing our phage buffer in some way?? I'm really glad I found this thread today because I was beginning to think I'd really messed something up. Amanda Edited 09 Dec, 2017 00:16 46Kb

Link to this post | posted 09 Dec, 2017 00:05

amandabbraley

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

Funny you should ask this, Dan. This is my winter break project! We had awful DNA degradation this semester! I was going to pull out old stocks of our nucleases and our old protocol and give it a try against the "new" nuclease mix and protocol.

Looking at the shockingly high proportion of smeared restriction digests this week (as we're doing presentations), it suddenly occurred to me that maybe (just maybe) it was something about the MP Biomedicals™ Deoxyribonuclease I (from Fisher ICN10057520). We previously used Thermo Scientific™ Pierce™ DNase I (#89836)–as well as an EDTA step.

We also had problems with low titers and our microscopist at WSU mentioned how "bad" our samples looked compared to years of other samples we've sent her. We're wondering if our M. foliorum phages aren't very stable in phage buffer or if the PYCa media system is causing some chemical reactions that are changing our phage buffer in some way??

I'm really glad I found this thread today because I was beginning to think I'd really messed something up.
Amanda

Edited 09 Dec, 2017 00:16

Link to this post \| posted 09 Dec, 2017 00:10
amandabbraley	Elizabeth Rutledge Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use. Thank you, Libby Hi Libby…just a note about the syringe barrels: Promega assumes you're going to use a vacuum manifold to suck the solutions through (rather than have the students push…. We've been using Wizard Kits for over 5 years now for 100 phages per semester…so I now have an entire cabinet of syringe barrels (I buy syringes with plungers separately). If anyone would like them, let me know. I really wish Promega would just sell the resin and columns. But then again, we're looking to try other ways to clean up our DNA. Amanda

Link to this post | posted 09 Dec, 2017 00:10

amandabbraley

Elizabeth Rutledge
Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use.

Thank you,
Libby

Hi Libby…just a note about the syringe barrels: Promega assumes you're going to use a vacuum manifold to suck the solutions through (rather than have the students push… smile

. We've been using Wizard Kits for over 5 years now for 100 phages per semester…so I now have an entire cabinet of syringe barrels (I buy syringes with plungers separately). If anyone would like them, let me know. I really wish Promega would just sell the resin and columns. But then again, we're looking to try other ways to clean up our DNA.
Amanda

Link to this post \| posted 09 Dec, 2017 21:20
smolloy123	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, We experienced this although two things to point out. We used phenol chloroform extractions and did not use Promega kit, treated lysates with EDTA after DNAse and RNAse treatment. I would think that the phenol chloroform would do the trick although maybe we needed to do two extractions in a row? Also, our DNA degraded only in certain restriction endonuclease reactions and not all the reactions.

Link to this post | posted 09 Dec, 2017 21:20

smolloy123

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,

We experienced this although two things to point out. We used phenol chloroform extractions and did not use Promega kit, treated lysates with EDTA after DNAse and RNAse treatment. I would think that the phenol chloroform would do the trick although maybe we needed to do two extractions in a row?

Also, our DNA degraded only in certain restriction endonuclease reactions and not all the reactions.

Link to this post \| posted 10 Dec, 2017 22:10
mdgainey	Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

Link to this post | posted 10 Dec, 2017 22:10

mdgainey

Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

Recent Activity

Yield and degradation of DNA isolated by new protocol