Link to this post | posted 06 Feb, 2016 20:18

jross1025

Page 48 of the current Annotation guide makes reference to "typical" and "atypical" coding potential in the GeneMarkS output, which I believe is the heuristically generated version. What is the distinction here? In both LilDestine and Teardrop, the two types of coding potential appear to be largely, although not entirely, overlapping; in those areas of sequence containing atypical, but not typical, cp, the atypical regions are generally small.

Link to this post | posted 17 Nov, 2015 23:55
jross1025	I think I'm starting to see the light………thanks

Link to this post | posted 17 Nov, 2015 22:52
jross1025	I'm also not using the qiagen nucleases, instead we're using 9 microliters of the nuclease mix from the old protocol. Not sure why I picked 9, but I figured if in the old protocol 2ml of resin could inactivate 40 lambda it could inactivate less than a fourth of that.

Link to this post | posted 17 Nov, 2015 22:49
jross1025	Marianne, the thing is that our yield problem is not across the board, and seems to be titer-related. I would think if it were a nuclease issue then the young lady with the e10 titer would be just as much affected as anybody, if not moreso. Plus I distinctly remember reading in Welkin's version of the protocol not to go above e11.

Link to this post | posted 17 Nov, 2015 02:10

jross1025

We did the new DNA purification protocol (i.e. no PEG) with five students Thursday and results were kind of mixed, which by itself I can totally live with given that we normally got mixed results with the old prep anyway and mixed results that only take one day to get beats basically the same thing that takes two days plus requiring a gunky reagent! But here's my question: At really high titers (ten to the tenth and above) do you all recommend that we really and truly dilute all the way down to say the middle of the ten to the ninth range? The best result we got Thursday was from a young lady whose phage titered out in the e10 range: she was able to make about 15 micrograms Then there were two in about the e11 range who made about 8 or 9 micrograms. The two who were in about the e12 range made rather little: a total of maybe five or six micrograms, tops. Nobody had any notable quantity of DNA in their second or third tubes (all per nanodrop). The e10 sample we used straight; e11 we diluted 1/1; e12 we diluted 1/5. I did this based on a piece of advice I got several years ago relative to the old protocol from Lu Barker, who advised against diluting anything beyond about 1/5. Any advice?

Link to this post | posted 06 Nov, 2015 19:32

jross1025

Thanks Deb (and Kevin), the new protocol looks interesting, however I have a minor question and a major question: Minor question: What is the "precipitate" in the resin(?) that the protocol refers to? I never knew it had any, I mean it has resin beads, but those aren't gonna dissolve in anything, and the bottle is sort of opaque anyway so I'm not quite sure I know what I'm supposed to be seeing dissolving, if that makes any sense. Major question: What about the kids with <5e9 titers? Use the PEG procedure? "Cheat" by centrifugally concentrating (i.e. pellet like you do for EM, and resuspend at lower volume therefore higher titer?)

Link to this post | posted 05 Nov, 2015 22:49

jross1025

I really need some advice. Tried my hand at making phage precipitating soln last night, got the salt dissolved fine, started to get the PEG into soln. But I can't really get it clarified or even completely dissolved (I think). I'm using the quantities given in the resource guide (19.3 g salt, 30 g PEG 8000, water ultimately to 100ml starting with 60).I tried alternating microwave heating and stirring on a hotplate, then I let it stir without heat for 18 hours or so and it's still opaque. I'm using some that's really old right now–it looks fine, but eventually I'm going to run out of it. It worked when it was new, and I have a hard time believing that anything really happens to this stuff. But again I'm eventually gonna run out one way or another. I have some that's newer but something happened to it in shipment, the bottle wasn't sealed well or something so I've never entirely trusted it. Any advice?