Marianne Junck, a Biomedical Sciences Major, joined the lab at the beginning of this semester. She will be assisting with studies of intraoral feeding and microdialysis, and is already off to a great start. Welcome Marianne!
Monthly Archive for January, 2006
Hi Rita,
Please let me know if there is anything you would like to add or change to the following. Especially important if the timeline for goals needs to be changed.
Since you’ll be gone on the 10th, let’s set our next meeting time on Thursday the 16th? Let’s go to lunch, I’ll buy.
Meeting 1/27/06 3:15-4:45 (!) Claire and Rita
1. Lots of goals set at our last meeting were met, and some progress on the PCR. The quantification is taking longer than expected because of technical problems. New goals for the next 2 weeks are:
>> a). Test out the spectrophotometer next door next week.
>> b). Optimizing experiments for combined analysis of increased annealing temperature and non-hot start with increased DNA concentration. The timeline for this will be 4 PCRs per week – running 2 a day, twice. The first two temperatures will be 61 and 64. We’ll determine the second two depending on the outcome of the first set.
i. For annealing, you’ll run 3 samples for each genotype – for each of these the samples will be from 2 different subjects, and one of those subjects will be run in duplicate (for a total of 9 samples across the 3 genotypes). Also run a blank.
ii. For additional, simultaneous analysis of non-hot start, you’ll run one of each genotype and a blank. Samples will be run with double concentration of DNA.
We decided that we will not pursue research into other factors affecting stringency until we’ve obtained these results.
2. We talked about handing over primary responsibility for the DD colony to you. This will include:
>> We’ll set up new breeding cages together on Tuesday 1/31 at 2:00.
>> You’re going to order or otherwise obtain a copy of Silver’s book, Mouse Genetics. Place and order / loan request next week.
>> We also talked about having you begin to create a document (or documents) describing our colony protocols. I’d like to look at your progress on this before our next meeting – so maybe send it to me on Monday or Tuesday (13/14th). I want you to have developed this further by the following meeting (~4-5 weeks from today).
3. Lab safety
Boxes for glass, tips (under-counter and counter-top). Lab coats. First Aid kit. Look into eye wash.
4. Misc.
OK – thanks!
Claire
Hi Rita,
So – here’s a little intro: I’ll try to provide a mtg summary like this when we sit down to talk, to make sure we stay on the same page. It helps me and I hope it will also be helpful for you. The format is somewhat abbreviated (thank goodness) – so when you see a “>>” that’s a “to do”. The rest is summary. In future, please respond within 2 weekdays if/when you see I’ve made an error, left something out, or you want to add something.
I *don’t* mean the “>>” to be Draconian. Usually we’ll have talked about the timeline – but – if, on consideration, a proposed deadline is not something you think you can meet given other demands on your time, please let me know. It’s much better to let me know ahead of time than to move back the deadline at the last moment.
If everything looks alright to you, there’s no need to reply.
Meeting summary 1/17/06: 2:15 – 3:00. Claire and Rita.
COLONY MANAGEMENT
I’m glad to know that our PCR for progeny from established breeders is in the ballpark for expected frequencies (!). Out of 64 pups born, that would be 12 DD, and 21 with only one locus (broken down to 13 Th-, 475 band, and 8 DbhTH, 575 band).
>> Tomorrow, I’d like to know how many HET breeders we have identified from the 64 progeny, and how many WT. For the HETs, email or print out a sheet like we discussed with cage, homecage, ET, DOB and sex.
>> Add five columns for genotype to your excel management spreadsheet – one each for WT (no bands); Th- (475); DbhTH (575); HET (475 and 575) and DD. When you get PCR results, put a 1 in the column for the correct genotype. Also, please add a column for “fate” – i.e., “breeder in cage X” or “used in experiment Y”
OPTIMIZING PCR
HOT START TEST
Also good to know that the PCR is working at a lower total reaction mix. (! excellent !) We discussed optimizing, especially to eliminate the 600+ bp band(s) and to determine what’s up with primer dimers.
>>This week, I’d like you to run a PCR to compare hot start with no hot start. Pick 5 known samples, one giving each predicted band pattern: WT (none); Th- (475); DbhTH (575); and two HET samples (475 and 575). When you run these, please run, in triplicate, a reaction complete mix with everything EXCEPT DNA added – under both hot start and non-hot start conditions (i.e., 6 total tubes). This second condition should tell us about primer dimers, and might tell us about non-predicted bands or contamination.
DNA QUANTIFICATION
We also talked about determining DNA content from our extractions. Turns out I needed to email April Harkins again today. She is an Assistant Professor in Clinical Lab Sciences. I asked her if she thought she would be a good resource for you – if she does, I’ll forward her contact info to you as soon as I hear back. Here’s the timeline I’d like to set for the DNA quantification project – since some of these goals are of intermediate length – I understand they are somewhat flexible. Let’s talk about whether we can meet the next goal when we get through them in order. (Hopefully, we’ll be ahead of schedule, even!):
>> By the end of this week (so, Thursday afternoon), send me a 1-2 paragraph summary of what you’ve learned about methods for quantifying nucleic acids r.e. DNA extraction / PCR. Spectrophotometry is how I’ve always done it.
>> By the end of the following week (1/27), let’s decide on a method to quantify the DNA we have now. I know the best solution may depend on the cooperation of / communication with others – but we want to have a good idea of our options by this time. I would like you to, if possible, come to the table with at least two different strategies (that may be differentiated as simply as “use machine in lab B instead of machine in lab A”).
>> If all goes well, we ought to be able to quantify our DNA the following week, by the end of the month.
>> Based on those numbers, run a PCR (or more than one if needed) with levels of DNA across samples normalized – to see if this improves our results. I’d like this goal to be reached by Feb 10.
RESEARCH STRINGENCY
We talked about looking into how our conditions influence the stringency of our PCR reactions. Temperature is a good one, and there’s also [Mg2+], [salts], etc.
>> By next Tuesday (1/24), run a PCR with our known samples with an altered (higher) annealing temperature. You might want to wait for the results of the hot start experiment to determine which method you’d like to use in future.
>> By next Wednesday (1/25), send me a 1-2 paragraph summary of what you’ve learned about how conditions affect stringency. Include with this a description of 2-3 (or more, if you like) specific experiments you’d like to run to try to increase the stringency of our reactions.
MISC:
>> Start a consolidated lab binder for ordering, received items. With an index at the front giving a single, non-repeating number to each order. Thanks!!
I brought you a copy of the department phone list.
You talked about wanting to visualize DNA without EtBr – I asked Chris in Behnam’s lab, he didn’t know of a way. I’m leaving that one to you, but keep me posted if you find anything.
>> Great job finding those labeling pens! Can you run a test on some off the EtBr surfaces (glassware, plastics, etc) to see if that will work? If so, label all that stuff with “EtBr” and a caution symbol – like that triangle with an exclamation mark inside it.
I’ve added backup capability for the lab website to my list of stuff to do with that. That’s a very good suggestion! Thanks. I probably won’t get to that until next month, FYI. I also plan to add some of our protocols there, in future. If you have any additional suggestions, let me know. For now, let’s backup onto the flash drive and every two weeks onto a CD. I’ll put some CDs into the drawer in the lab desk today – and put a backup reminder on the lab web calendar (adding functionality to that is also on my to do list).
The door construction has been scheduled for JAN 31. That’s evidently official according KBS Construction Project Superintendent Jim Babe Jr (414) 810-9707. I changed the lab calendar accordingly – and next time we meet we’ll finalize our plans for dealing with the chaos that will no doubt ensue.
Finally, this is just a head’s up. In a month or so, I’m going to want to sit down and look at the PCR records and decide whether we need to make any changes to our record-keeping. Like, maybe we need to alter the data blank or get a better printer or … I just wanted you to know that and let you know that any thought you have about it would be good to bring up at that time.
>> Let’s meet again at the end of next week to see how we’re doing – let me know if Friday, 1/27 at 2:00 will work for you? This time I’ll bring some fruit or crackers and cheese and we can make a pot of tea or something. Let me know if I’m crazy and I should bring chocolate instead.
I just want to say again that I’m very glad to have your help and thanks for all of your efforts – I feel that we’ve made alot of good progress and I really appreciate your hard work and resourcefulness!
Cheers,
Claire