Category Archives: Protocols

Lab Website Organization

This website is set up to have primarily dynamic content. There are a few static pages, but most of the content is in the form of “posts” that are organized into “categories”. Lab-members aim to post updates at least once a semester on what they’re up to. We’ve also started a biweekly undergrad journal club under the “What we’re reading” category. There are also posts on funded projects, general cool science, and protocols that we’ve found handy.

Software Carpentry Workshop

Maren, Roxanne, & Ellie attended the hpcc swc bootcamp. Thanks instructors!

Maren’s notes from the bootcamp are posted here.

From the organizers:
We hope you enjoyed the Software Carpentry bootcamp this week and picked up a few new skills to speed up your research workflow.
If you’re still unsure how to integrate any of these tools or skills into your workflow, we’d be happy to meet with you to discuss your specific needs and help you get started. (Note: The longer you wait to integrate these skills into your workflow, the less likely you are to do it, so it’s best to start now.)
Respond to this email if you’d like to schedule consultation with us. As always, the only cost is your time and effort.
MSU’s ICER department also offers weekly office hours and can provide similar consultation on a weekly basis.
We’d also like you point out that Software Carpentry has free online materials (video lectures and notes) for all of the material we covered in the workshop (and much more).
Finally, if you’d like to get involved with Software Carpentry (or know someone that might want to), take a look at SWC’s Get Involved page. We’re always looking for more helpers and instructors at bootcamps, new places to run bootcamps, and new contributors to help us improve our teaching material.
Cheers,
MSU Software Carpentry team

How To: Post on the Lab Website

  1. Login
  2. Go to the “Dashboard”
  3. Select “Posts” / “Add New”
  4. What You See Is What You Get
  5. “Add Media” / “Upload Files”
  6. “Categories” — select appropriate categories so that navigation remains a snap
  7. “Preview” — proofread!!!
  8. “Publish”

Making ape work on Mac OSX 10.8

ape 3.0.7 on Mac OSX 10.8 has some problems reading fasta files! read.dna doesn’t see “G” (???) and line endings are messed up.

From the internet (http://comments.gmane.org/gmane.comp.lang.r.phylo/2509):

Feb 6, 2013

“Hi Rupert, The Mac version of ape on CRAN is not correct and has a few bugs particularly in read.dna(). Another problem is that “G” is not read (but “g” is). This does not affect the Windows version nor the sources. A solution for Mac users is re-install from the sources. Another one is to use the code of read.dna() from a previous release of ape.   I’m waiting for other tests and feed-back before releasing ape 3.0-8 (within one or two weeks).

Best, Emmanuel”

 Solution: install from source:

1. Install Xcode (from Mac App store, running OSX 10.8.2)

2. In Xcode, under “Preferences”, “Downloads”, select “Command Line Tools” install button

3. Still need gfortran, go to http://hpc.sourceforge.net/ and download http://prdownloads.sourceforge.net/hpc/gfortran-mlion.tar.gz?download (for Mountain Lion only).

In Terminal, cd to the Download folder:

gunzip gfortran-mlion.tar.gz

sudo tar -xvf gfortran-mlion.tar -C /.

4. In R Installer, select CRAN (sources) and install ape 3.0.8

5. It works!

Lab Safety

SAFETY TRAINING

Please print out your certificates of completion for the lab safety binder. ALL lab members must complete:

If you will be working in the greenhouse, you must also complete pesticide safety training (schedule through Maren or Jimmy with the greenhouse staff).

References:

Biosafety manual

Bacterial DNA extraction

1. In a biosafety cabinet, transfer 1000uL of liquid bacterial culture from a 15mL conical tube to a microcentrifuge tube. Return the conical tube to 4˚C.
2. Spin the microcentrifuge tube for 5 minutes at 10,000g to pellet the bacterial cells. Discard supernatant.
3. Resuspend the pellet in 150uL Tissue & Cell Lysis Solution (Epicentre), and add 10uL Proteinase K at 20 mg/mL (Qiagen). Incubate at 55˚C for 30 minutes.
4. Add 2 uL of RNase A at 5 ug/uL (Epicentre), and incubate at 37˚C for 60 minutes, followed by 5 minutes on ice.
5. Add 75uL MPC Protein Precipitation Reagent (Epicentre) and vortex for 10 seconds. Centrifuge at 12,000g for 10 minutes. Note: following this centrifugation, the protein pellet will be entirely transparent. Be sure to orient the tubes in the centrifuge such that the pellet will be in a predictable location.
6. Transfer supernatant to a fresh microcentrifuge tube and add 250uL isopropanol to the supernatant.
7. Mix tubes by inverting 10 times, and incubate at room temperature for 20 minutes.
8. Centrifuge at 12,000g at 4˚C for 10 minutes. Discard supernatant without disturbing the pellet.
9. Add 500uL 70% ethanol and centrifuge at 12,000g at 4˚C for 5 minutes. Discard supernatant.
10. Repeat step 9.
11. Remove as much ethanol from the tube and dry the tube with the cap open on the bench for 20 minutes, or until entirely dry.
12. Resuspend the DNA pellet with 50 uL TE buffer. Incubate for 5 minutes at 55˚C to resuspend the pellet.
13. Vortex briefly and spin down. Store at -20˚C.

PCR (Polymerase Chain Reaction) for 2 Different Primers

Procedure:

1) Prepare 1 Master mix to make sure reagents in both primer reactions are homogeneous.

2)  Take reagents out of freezer and let them thaw. (buffer, DNTPs, Taq, DI Water, and 2  different primers).

3)  Get ice bucket for reagents while not in use.

3)  Add DI Water into tubes first, then add reagents. (DO NOT ADD PRIMERS YET)

4) Split Master mix into two (111 microliters) and put into two separate tubes.

5) Add 7.9 microliters of one primer to one tube and add 7.9 microliters of other primer to the other tube.

6) Pre-plan in notebook well-plate setup

7) Add DNA to well-plates.

8) Centifuge well-plates (DO NOT FORGET TO BALANCE)

9) Place in thermocycler to begin PCR.

Culturing Bacteria into Liquid Media

1. Clean and sanitize the bio hood with 70% EtOH. Clean gloves and all materials that will be entering the hood.

2. Remove the pipette from packaging and attach to dispenser.

3. Label all test tubes with bacteria type, colony letter, your initials and the date.

4. Place 10 mL of the liquid media into each test tube.

5. Remove the parafilm from the petri dish and open lid.

6. Use a inoculating loop to select a single colony of the bacteria and place the loop head into the solution to dispense. make sure to rinse throughly.

7. reseal the petri dish and mark the colony’s location with the colony letter.

8. seal all test tubes. place the samples into the incubator for about 24hours.

****with each batch of bacteria cultures, there must be a control negative tube that contains the liquid media but no bacteria sample. this is to ensure no contamination has occurred.

LB Media Protocol

FOR 600mL OF LB BROTH MEDIA:

1. Weigh out 6 grams of Tryptone

2. Weigh out 3 grams of yeast extract

3. Weigh out 6 grams of NaCl

(3.5). FOR LB AGAR MEDIA: Weigh out 9 grams of agar

4. Combine all of the necessary substances into a 1L bottle

5. Measure out 600mL of distilled water into a graduated cylinder and pour into the bottle

6. Tighten the cap, Mix, Perform the quarter turn for ventilation, tape the cap to the bottle with autoclaving tape, and label the contents of the bottle; the bottle is now ready for autoclaving.

 

 

Extracting DNA from Leaf

Procedure:

1) Take ~0.5g of leaf matter and place in 15mL tubes

2) Add 1mL of 0.2M NaOH to the tube

3) Boil for 1 minute

4) Add 1mL 0.2M HCl and 0.5mL of 0.5M Tris 8.0 plus 25% Tween

5) Mix and cover the tube (allow for ventilation)

6) Boil for 2 minutes

7) Remove all liquid

8) Place tissue in mortar & pestle.  Add 0.5mL 10mM Tris 8.0.  Grind

9)  Add 1mL of 10mMTris

10) Use 2 mL of the liquid for PCR