Link to this post | posted 02 Mar, 2016 18:30

debbie

Keith, I asked Dr. Lawrence about this and here is his response: "Yes, DNA Master will wait until the BLAST server send replies; the delays are due to the failures of the BLAST server to return results in a timely fashion. Outside of abandoning searches and resubmitting the data - which they explicitly tell you not to do as it simply overloads the servers - there it nothing one can do. Their policy does ignore the possibility that the server may never return the results, or return them after an inordinate amount of time." Not very satisfying, but may help us to understand. I blasted a genome yesterday that produced the usual errors and took 8 hours to complete, but it did finish.

Link to this post | posted 29 Feb, 2016 23:43
debbie	Success! I blasted an entire phage genome (52KB) and finished the blast. The blast log details that the blast finished in about an hour, but it could not retrieve all of the blast data for about 3 additional hours. However, the blast did finish and it is looking good so far!

Link to this post | posted 29 Feb, 2016 19:34
debbie	Hi all! I was just able to blast a gene (and repeated by Dan) in DNA Master. The Integer Overflow error is gone and my posted wait time was 31 seconds, with the blast data returned in about 1 1/2 minutes. I just started to Blast an entire genome and will keep you posted as to how that goes! We are back in business!

Link to this post | posted 27 Feb, 2016 22:15
debbie	Hi all. It is not a DNA Master issue, but rather an NCBI Blast error. Note when you blast, it returns an error message "integer overflow" and tells you that the wait time is a couple of years (mine said 54956368 seconds). We have been in contact with NCBI and they "are working on it". in the meantime, you can blast one gene at a time and record your results. We will keep you posted.

Link to this post | posted 11 Feb, 2016 22:18
debbie	Greg, Though it is always helpful to know the whole context, I think you are asking about what GeneMark calls a possible frameshift. It has identified some sort of ribosomal slippage. Know that we do not call any frameshifts except for the programmed frameshift found in the tail assembly chaperones found upstream of the tape measure protein. Make sense?

Link to this post | posted 27 Jan, 2016 20:50

debbie

Tammy, The best answer is all of the above! I use GeneMarkS and GeneMark with Arthrobacter FB24. Whatever you use, record that in your cover sheet. The one directed at self is the same info as what comes in through the auto-annotation, just in picture form. So it is not additional info. Both GeneMark against self and against a host are helpful and informative. Blasting against phagesDB (and Phamerator - which is the same data) is just identifying what we already know. Now that we have >45 Arthrobacter phage genomes finalized and in GenBank, you should find more helpful info there. However, I will always say you should run an NCBI BLast because there is always new data being posted there. Likewise, HHPred should be run with the 3 databases pbd70_, pfamA_, and tigrfam_. Again new data is showing up there regularly and we are able to identify more functions to our genes than ever before.

Link to this post | posted 17 Dec, 2015 21:02

debbie

Recently I was asked via email to give my best "tricks' to avoid clumpy smeg. After my suggestions worked, I was asked if I could post them, so here they are. Here are my recommendations: Streak out the smeg that you have – frozen if possible. Once you have colonies (in 3 -4 days) that you can recognize as smeg - flat, wrinkled, dry, and tan – more yellow if you are growing on LB – sub into small aliquots (2-4 mls) of growth media with Tween into test tubes. Final Tween concentration is recommended at 0.05%, but I would increase that 5x. Inoculate with a small inoculum from a single smeg colony – the smaller the better. Place on a shaker and shake vigorously for 2 days. Vortex and then check this culture. Is it homogenous and creamy or do you have clumps? I set up 3- 5 tubes this way. Invariably, one doesn’t grow, one is clumpy and one is perfect – smooth and creamy. This is my started culture for the entire semester. To set up my big cultures – and I mean big – because I set up 1-2L of smeg at a time. I add all of my media ingredients to a flask and then add 1 drop of the starter culture. It will take at least 3 days to grow this up to a saturated culture, but if I need smeg in the meantime, I remove what I need. (always putting the flask back on the shaker). I also avoid allowing others to enter my flasks of smeg to avoid contamination. I also aliquot by day, meaning I only permit one entry into that flask/day. Eventually, contamination will occur, so drop the number of occurrences. If your students have phage, but just can’t purify and amplify – you can try adding Tween to all of your liquid cultures – you will know if the Tween is problem or not. It usually is not. You can’t do that if you have not found any phages. Short list of things to do: Start your ‘starter’ cultures from single colonies that you recognize as smeg. Use Tween (lots of it) in your starter cultures Grow big volumes so you don’t have to keep growing it. You could grow ALL that you need for the remainder of the term right now (if you have flasks that are big enough). Keep the flasks on the shaker until you are sure the culture is not growing any more (has reached saturation). Edited 17 Dec, 2015 21:03

Link to this post | posted 06 Nov, 2015 20:04

debbie

Joe, Minor question: The precipitate is the beads. Some folks don't realize that the resin needs to be well(evenly) suspended. Precipitate is probably not the right word. Major question: I would try anything but PEG precipitating. The literature says that you lose about 1/2 of the phage concentration when you do it. So if your lysate is close to 5 x 10^9, then I would just try an extraction. If it is not, concentrate by centrifugation and resuspend in a smaller volume. (and that is not cheating!)

Link to this post | posted 06 Nov, 2015 18:49
debbie	Hi Joe, I heard that you spoke with Kevin yesterday about this. I am just adding a link to the protocol that is already on seaphages.org. http://seaphages.org/media/docs/NewDNAProtocol.pdf

Link to this post | posted 15 Sep, 2015 17:41
debbie	so it sounds like you are testing your post-enrichment plates by putting 50 ul of sample into smeg and plating. Are you also doing spot testing? Sometimes 50 ul of enriched samples can so 'blow-out' the cells on a plate, you see no plaques. If the plaques are turbid, you will see lighter confluent growth of cells, so it can be quite confusing. Just thinking.