SEA-PHAGES | All posts created by amandabbraley

Link to this post \| posted 20 May, 2019 23:07
amandabbraley	I also just grabbed some screenshots from student posters this semester to show the range of results we see with M. foliorum phages. There are 8 different phages shown here. 381Kb

Posted in: Phage Biology → DNA Smear

Link to this post \| posted 20 May, 2019 23:06
amandabbraley	We've had quite a bit of smeary DNA from our M. foliorum phages over the last two years. We adopted the Proteinase K/EDTA steps and investigated a few other modifications, including changing some of the temperatures used during the nuclease steps. Our thoughts were that the capsids of some phages were very "unstable" and didn't protect the phage DNA during the extraction protocol. I've attached the poster our students took to the SEA SYMPOSIUM last summer about this issue. 1.43Mb

Link to this post | posted 20 May, 2019 23:06

amandabbraley

We've had quite a bit of smeary DNA from our M. foliorum phages over the last two years. We adopted the Proteinase K/EDTA steps and investigated a few other modifications, including changing some of the temperatures used during the nuclease steps. Our thoughts were that the capsids of some phages were very "unstable" and didn't protect the phage DNA during the extraction protocol. I've attached the poster our students took to the SEA SYMPOSIUM last summer about this issue.

Posted in: Phage Biology → DNA Smear

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

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

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

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

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