Rohwer Wegley-Kelly Lab

Biol567L Spring 2015

Announcements

Date Announcement
2015-08-25

People are making good progress with their plates.

In accordance with the syllabus, please make sure that you have your plates made by the end of Wednesday's class.

Regards,

Ben.

File Downloads

Date Title Description
2015-12-02Th End is Nigh!

Attached is a guide to the completion of 567L.

Next Monday and Wednesday (if we need it), will be group presentations. I have put a schedule at the bottom of the attached document. People currently scheduled for Wednesday need to be prepared on Monday in case we have time to get to you.

I will update grades by the time class meets on Monday.

Everyone has done quite well. Expectations are high; keep up the good work!

b

 

2015-11-03Come to class next Monday and give a 5 min talk on your progress

Everyone needs to come and give the class a five minute presenation on their project next Monday. It will be scored out of 10 points.

You need to tell us your:

Hypothesis

Areas of success

Areas of difficulty

Revised timeline

b

 

2015-11-03Do not come and find me on LSN302; text me and I will find you as soon as I can

b

2015-11-03Do not come and find me on LSN302; text me and I will find you as soon as I can

b

2015-10-28grades to date

Please submit all weekly updates to Forest and I with the subject line

567L weekly goals <Disease name(s)> Date

Thank you.

2015-10-23Send me a list of things you need to buy; come discuss list on Monday

Hi

Please send me a list of the things your group needs but have not been able to get yet before Monday.

Come down on Monday to discuss the list with me. I'll be in the lab starting at 1pm and again at 4pm.

b

 

2015-10-19Safety training today 1pm and 4pm

Please attend a safety training session today at either 1pm or 4pm in the classroom.

2015-10-12grades to date - updated with presentation feedback scores

up to date as of Mon 12th 5 pm.

2015-10-12This week - practical and quiz today, bioinformatic homework due Wednesday, 16S sequence analysis due Monday

Assessment over the next week:

Today - practical and quiz. Provide proof you successfully completed the task set on Wednesday.

Wednesday - submit your bioinformatics homework to my email.

Next Monday - bring a printed copy of your 16S analysis report (11 points total). Rubric is:

2 pages with the canonical format Introduction (1 pts), Methods (3 pts), Results (3 pts), Discussion (1 pt), References (2 pts). Make your references meaningful.

Any pages beyond the set two pages will not be graded.

b

 

2015-10-09Practical next Monday, Next Round of Proposals next Wednesday

Next Monday we will have the practical exam.

You will be required to take a quiz on all aspects of the class to date, with a particular emphasis on solutions and dilutions. You will also take a practical assignement such as making and running a gel, etc.

As ever, you can use whatever resources you would like to complete these tasks.

On Wednesday we will do another round of proposal talks for those people who have not been signed off to proceed with their projects.

b

2015-10-08Industry Job Description

Please find attached a job description for a technician position in industry. Forest thought you all might find it interesting to see what industry is looking for in potential employees.

b

2015-10-07Sequences are back!

Sequences for Jurri, Laura, Zac, Rebecca, Donny, Kevin, Courtney, Brian, John, Gianna, and Kyle are back!

Everyone else, get me your DNA by the end of class today. Recipe is on the board.

:D 

2015-09-28For Today - Gels

Hello Students,

Please come to class ready to show Forest and I your gels with plasmids in them.

We require: Ladder, plasmid. As you should have a ton of plasmid DNA (far more than the 100ng required to make a band on the gel), no PCR amplification, positive controls, etc are necessary.

There will also be a quiz.

b

2015-09-28Sending DNA off for sequencing

We will be sending your DNA off for sequencing by a company called Retrogen.

They require you to mix up 100 ng of template DNA (i.e. quantified) at a concentration of 20 ng/ul, as well as 10 umol of primer (at 10 umol/ul), and 5 ul of sigma water (12 ul total rxn). 

They will amplify your DNA and Sanger Sequence it.

Please also provide me with your sample details when you give it to me.

b

2015-09-28Bioinformatics

Please find attached the class Bioinformatics BLAST take home. Due Oct 7th, the day of the practical.

:D

2015-09-28Bioinformatics Update

Also try BLASTing the sequence in the Bioinformatics take home document here at http://rdp.cme.msu.edu/seqmatch/seqmatch_intro.jsp

:D

2015-09-28Proposals

Guide to your powerpoint proposal. Don't miss any of the elements explicitly listed below.

Make sure your question revolves around biochemistry, cell and molecular biology. Do not feel limited in what techniques you can pursue. You cannot use a hadron collider, but we have never been unable to accommodate students before. Things like cell culture, sequence a genome, proteomics with 2-D gels).

Background - 1 slide - the ideas or observations that you have made that are leading to you asking your question.

Hypothesis - 1 slide - make sure your hypothesis is non-trivial (i.e. not obviously true), explicit, precise (vague now means miserable later), and falsifiable (how will you know if the hypothesis is true unless you make clear predictions that could be wrong?).

Remember: being wrong is the best thing that could ever happen to you!!

Methods - 1 or 2 slides - outline the approaches you will be using, controls and protocols etc, required equipment, budget. Think this through.

Expected Results - 1 or 2 slides - show us a diagram of your fantasy data, with controls in it. What graphs would you get if everything went well? If this is easy, it likely means that your hypothesis is well concieved and precise. If it is not, perhaps your hypothesis is too vague and you need to think things through more...

Timeline - 1 slide - outline what you think you can achieve over the rest of the class. Put milestones in that you/we can use to check your progress and know that you need help.

Next class people in labs (e.g. Coutney, Tracy, Juri) will present their proposals as a guide to everyone else. We will discuss ideas you all have afterward. Everyone else goes next Monday. Don't leave this until the weekend. Poorly concieved talks are miserable.

b

 

2015-09-11Update for the Week of Sept 14th: PCR

Dear Students

A few things.

1) People have started stealing other people's reagents. That is a bad idea because you have no idea if the owner contaminated their reagents, if they mishandled their reagents, etc. i.e. you have no idea what you are ACTUALLY stealing.

2) People have been taking their reagents out of the freezer, and leaving them out on the desk as they work. You will ruin your reagents this way. PCR is set up on ice. Taq goes from the freezer to ice. It does not do room temperature. 

3) In molecular biology, precision with small amounts beats sloppiness and desperation with large amounts. You will not succeed with twice the volume if you were going to fail with the correct volume. Molecular biology is a science. It is refined. Two positive controls with the same DNA and master mix is no better than one. What would you comclude if one worked and the other didn't? Why would you waste your reagents?

4) I have provided you all with individual aliquots of all of the reagents you needed for ~30 reactions. Some groups have burned through all of their reagents already, despite the improbability that they have done that many. Make sure you are pipetting correctly, or else the PCR will never work. Precision and accuracy are key.

5) When you are troubleshooting your PCR, use 25ul reaction volumes per tube. 50ul will just use twice as much reagent. Until you get your positive and negative controls to work, consider doing PCRs with just those two reactions.

6) Tracy has aliquoted you all new DNAse and DNAse buffer. Brandon has aliquoted you new water. Rebecca has aliquoted you new dNTPs and Dye. Take one aliquot of each. You will have enough reagents for another ~50 reactions. If you run out, you need to send me your PCR table and list of what you have run so we can figure out what is going on. Then aliquot it out more yourself. Do NOT have all 50ul of Taq in one aliqout that you then freeze-thaw every time you make a new PCR. Make ~10ul aliquots so that you only take out from the freezer what you need at any time. This will save you a lot of pain and radically decrease your chances of 1) ruining your reagents through mishandling, and 2) contaminating your whole supply of reagents.

7) The Taq needs to DNAsed. Add 1ul of the aliquot you've taken to the 100ul aliquot of Taq you've taken. Add the buffer at the correct concentration. Mix it. Incubate it for ~3hr at ~37C. Don't shake it. It's an enzyme. An expensive enzyme. You won't get more of it. Then incubate it at 65C for 15 min. Then aliquot it out. Run a PCR just with negative and positive controls. Figure out why you are doing this...

8) KEEP ALL ETHIDIUM BROMIDE-CONTAMINATED EQUIPMENT WITHIN THE RED TAPED AREA IN THE COMMON ROOM. OTHERWISE YOU EXPOSE OTHER PEOPLE UNKNOWLINGLY TO THE POISON. NOT COOL.

9) If you can demonstrate that your PCR has worked and show a picture of your gel and explain it to Steven, he will give you the reagents to continue to cloning. DO ONE REACTION. USE 1ul PER REACTION. IF YOU TAKE MORE THAN YOUR SHARE, SOMEONE ELSE CANNOT DO THEIR WORK. Again, we will not get more.

b

2015-09-10Grades Uploaded

Here are the grades to date. 

On future quizzes please put your codenames instead of your real names, and I will add your grades to this spreadsheet.

b

2015-09-07Gel Reagents available

People are starting to run gels so they can find out if they have DNA from their PCRs.

To enable this, I will put some ladder (figure out how much you need, as this should be enough for a lot of gels!) in the classroom freezer and some Ethidium Bromide in the gel area of the common space (also figure out how much you need, and remember, if your gel is at all purple you have way too much EtBr).

b

 

2015-08-24Syllabus for Fall 2015 567L
2015-08-24BAM paper

This is the paper that established the Bacteriophage Adherence to Mucus (BAM) model of non-host derived immunity.

2015-02-18T7 Cloning Manual
2015-02-03Taq Isolation
2015-02-03Book suggestion

If you are not completely comfortable with making dilutions and such, then go get Stephenson's Calculations for Molecular Biology and Biotechnology.

2015-01-27T7 Cloning manual

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Rohwer Laboratory, San Diego State University.

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Lab Info
North Life Sciences Biology Bldg
San Diego State University
619-594-1336

PDF: Lab Directions
Map: 32.777818, -117.071543

Forest Rohwer, PhD
Professor of Biology
SDSU Dept. of Biology

frohwer@gmail.com