How-to

See also: Protocols

Computer

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Notebook/Data Management Best Practices

1. Do not upload files directly to the wiki.
2. Provide links (full url) to ALL data files and images.
3. Dropbox should only be used for temporary storage and movement of files.

Accessing data server - The Eagle

Uploading iWeb Page to UW Server

Direct connections from iWeb to server does not seem to work. Therefore, it is a two step process; 1) Save locally and 2) Upload files to server using SFTP client such as Fugu for Macs. If you have not done so, you will need to activate web services, complete details @ IT Connect
1. Download Fugu.
2. Connect to UW server. For students that is dante.u.washington.edu. screenshot
3. Your files need to be in the public_html folder so you can open that directory.
4. In iWeb, save site to local folder. I usually make tmp folder on desktop.
5. In Fugu you can locate the local directory then simply drag to public_html directory. screenshot'
6. That should be it. Just navigate to http://students.washington.edu/ your_uwnetid /

Configuring iWeb to Publish Web Page Updates to UW Server

1. Open iWeb and Fugu (see above for download link).
2. In Fugu, connect to UW server. For students that is dante.u.washington.edu
3. Double-click the public_html "directory". This is actually a shortcut to the physical location that your files are stored on the server.
4. Highlight one of your files in the current directory.
5. Click the "Info" button in the Fugu menu bar.
6. Copy the information next to the "Where:" category.
7. In iWeb, click on "Site" in the left-hand column.
8. Change "Publish To" to "FTP".
9. Enter the "Server Address" which is dante.u.washington.edu
10. "User Name" is your_uwnetid
11. "Password" is your UW password associated with your_uwnetid
12. "Directory/Path" paste in the information you copied in Step 7 above. At the end of this information add your_uwnetid
13. Change "Protocol" to "SFTP".
14. For "URL" enter http://students.washington.edu/your_uwnetid


15. Push the "Publish Site" button on the bottom menu bar of iWeb. This first time you publish will take some time to upload all your files. After that instance, iWeb will monitor which files have been changed and will only publish those files when you push the "Publish Site" button, which results in the site publishing being extremely fast.

Configure Skitch to Upload and Share Images to/from Eagle

1. Open Skitch Preferences.
2. Click on the "Sharing" tab/button.
3. Click on the "+" symbol at the bottom of the "Sharing Accounts" window.
4. Provide a name for the server (Eagle).
5. For "Type", choose "SFTP".
6. "Server": eagle.fish.washington.edu
7. "Port": 22
8. "User": Your Eagle username
9. "Password": Your Eagle password
10. "Directory": ~/web/your_folder_name/your_folder_name
11. "Base URL": http://eagle.fish.washington.edu/web/your_folder_name/your_folder_name

12/2/2013 - kubu4

Visitor/Guest Wireless Access

Go here.

Set Up Panasonic BL-C131 Wireless Webcam for Wireless Operation

1. Obtain a computer running a Windows OS and start the computer.
2. Obtain the webcam and check the switch underneath to make sure it is set to "wireless."
3. Obtain the Panasonic software disc (ask Steven or Sam for location) and insert into disc drive. Software should launch automatically (if not, open "My Computer", double-click on the CD drive icon and locate the "Setup.exe" file. Double-click on this file to launch the software).
4. Click on the "Search for Cameras" button.

Automatic Computer Backup

Note: The Lion operating system (OSX 10.7) will NOT work with Time Machine as described in this protocol.

Everyone in the lab needs to make sure your computers are backed up on a regular basis.

• Please use Mozy for critical materials- use this link to install Mozy on your personal computer, sign up for your own account with 2 GB of free storage. This will simply run in the background and is a nice complement to Time Machine.
• To use Time Machine to back up to a centralized server in the Lab.. In Finder, select Go, Connect to Server. Server address is afp://aquacul4.fish.washington.edu. Enter username and password as you would as if you were using any "lab" computer (ask someone if you cannot figure it out). Connect and select "Chickadee". In System Preferences, Select Time Machine, hit Select Disk and select Smelt. Under Options.. you can select stuff not to backup, however you are welcome to backup your entire computers.
• Dropbox is great because anything you have in Dropbox is automatically backed up.
• Crashplan is another excellent option that has a free scenario.
• Google Docs (Drive) is yet another option for backup.

8/2/2012 - SJW

Embed Pictures in Your Notebook

Hosting images on Eagle.
1. Transfer desired picture to your folder on the Eagle.
2. Visit said folder via eagle.fish.washington.edu
3. Navigate to your picture of interest.
4. Right-click on your picture and select "Copy link location..." Alternatively, click on the file. The file will open and be displayed in your browser. Copy the URL from this page (found in the address bar at the top of the browser).
5. Go to your Notebook and begin editing.
6. Click the "File" button in the Visual Editor bar.
7. Click the "External Image" tab.
8. Paste the link to your file in the space provided.
9. Click the "Load" button. A very small icon of your image will appear below the space where you pasted the link.
10. Double-click the very small icon of your image. This should return you to your notebook with the image in place.
11. To resize your image, click on the image and click-and-drag any of the 8 handle bars (black squares).

If this is not an option or to see 3 other ways to include images in your notebook check out this video:

- Flickr is another way to host images in your notebook. The lab has a profession account.

From a December 2010 Lab Meeting, Sam shows us how to embed about anything in a notebook- sr320 Jan 11, 2011

Edit a gel image

• Splash-up
• Flickr (cannot draw, but can include notes, ie box a band)
• Highlight w/ a little screen capture.
• Skitch!

Manage Large Notebooks (add Page Widgets)

If your notebook is starting to load slowly and/or edit slowly during editing, you should create smaller "working" notebooks and embed them in your main notebook. To do this, starting at the lab wiki page:

1. Click "New Page" (above the search bar on the wiki)
2. Name your new page and save. It cannot be the same name as your primary notebook (i.e. can't be "Sam's Notebook", since this page already exists with this name). It is a good idea to come up with a naming scheme that can be consistent for each time you create a new "working" notebook for embedding.
3. Go to your primary notebook by clicking on the link on the left-hand side of the wiki.
4. Click "EDIT" (upper right-hand corner of your notebook page, surrounded by white box) and set the cursor above your most recent text entry.
5. Click the "Widget" button on the editing bar above.
6. Click "Contents of a Wiki Page." (If you do not see this, you may have to select "Wikispaces" from your choices on the left hand side of the pop-up window).
7. In the "Page Name" box, start typing the name of the new page ("working" notebook) you created in Step #2. Click on the name.
8. Check the box next to "Editable."
9. Click the "Embed Wiki Page" button.
10. To edit your "working" notebook, click on the small word "Edit" at the top right corner of your notebook page. Do NOT click on the larger, all caps "EDIT" (surrounded by white box).
10/15/2010 SJW

Use the lab calendar

Post to the tumblelog

Analyze qPCR data Using PCR Miner

BioRad Opticon2 Raw Data Extraction
1. Open data file using Opticon 3 software
2.



BioRad CFX96 Raw Data Extraction - kubu4 - kubu4

1. Open data file using CFX Manager Software
2. Click the "Quantitation Data" tab.
3. Change the drop down menu to "RFU".
4. In the "Settings" menu, change "Analysis Mode" to "No Baseline Subtraction".
5. Right-click anywhere on the data and then "Export to Excel..." and save the Excel file.
6. Follow the Miner video tutorial below or the PCR Miner website to learn how to correctly format your data for PCR Miner Analysis.
7. After data is returned from PCR Miner, follow the video tutorial below and/or see the Example PCR Miner Analysis file (Excel).

• PCR Miner website

Video Tutorial on How to get Opticon data into Miner

Analys qPCR data - Simple and Crude

After you run your PCR and you get me or someone else to explain / analyze it,

you will be able to covert C(t)s to arbitrary expression values using the following formula.

Arbitrary expression value =10^(-(0.3012*Ct)+11.434)



More info:

Aggregate RSS Feeds

More Info: "Google Reader for Academics @ Bitesize Bio"

Search / Manage Literature

• More Info: "18 ways to improve your Pubmed Search @ Bitesize Bio"
• Do you want your google homepage or yahoo front page to have PUBMED search results for your topics of interest. Create an RSS feed at PUBMED and add this feature to your RSS reader. Now just like news and sports highlights you can get publication highlights with your favorite RSS reader. [video via Bioscreencast] - sr320 Sep 14, 2008
• I thought I would give some suggestions on where to go to look for what your looking for, if you are looking for something. Primarily I use Pubmed. Most genomic / molecular journals will be covered here. For more traditional fisheries articles, head over to ASFA: Aquatic Sciences and Fisheries Abstracts. For this one you need to be on the UW network. And what list would be complete without a product from Google, Google Scholar. What is great about this is not only can it be used anywhere, but you can set it up to automatically provide direct UW link. - sr320
• Papers
• Zotero

[ http://feeds.delicious.com/v2/rss/sr320/literature]

Submit Sequence to Genbank

• Bankit

Video Tutorial. Using Bankit

• Sequin

Bioinformatics

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Synonymous and non-synonymous SNP detection

How to identify synonymous and non-synonymous SNPs using CLC and without a reference genome Video Tutorial


Part 2

Find info on your favorite gene(s)/pathway(s)

See some great example of some that already have.

Resources:

• NCBI

Video Tutorial. How to quickly use BLAST to find out more about an EST

Blast tips [via NCBI]

• genomic_it (download)

Video Tutorial. How to use genomic_it [Hi-Res]

• iHOP
• Panther
• The Reactome Book: a textbook of biological pathways
• Gene Ontology

Video Tutorial. How to generate GO Pie Charts [Hi-Res]

• cGRASP (Salmonids only)
• KEGG

[ http://feeds.delicious.com/v2/rss/sr320/pathway?count=15]

Identify Intron Location

• Spidey

Video Tutorial . How to find Intron location in genes [Hi-Res]

• Splign

Design Primers

Tools available include

• NCBI primer Blast
• Geneious
• IDT
• BiBiServ GF2 (I have not tested) - sr320 Sep 14, 2008
• iCODEHOP - sr320 Feb 13, 2011

See also: post on approach for gene discovery in Vt

Video produced for FISH441

Align Sequences

• Geneious
• Blast
• NCBI BLINK

Video Tutorial: Sequence Alignments using BLINK and Geneious [Hi-Res]

Identify proteins from 2D gels

Lab

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Run an Agarose Gel

Select gel mold size to accommodate number of samples (including a ladder or two or three)

Measure an appropriate volume of 1x TAE (volumes listed below are for the Denville system):

• Small = ~75mL (50mL)
• Medium = ~100mL
• Large = ~175mL (150mL)

Add agarose, making sure not to contaminate (do not pour extra back in to bottle)
For a 1.3% gel (rather firm)

• 1g in 75 ml
• 2g in 150 ml

Add Ethedium Bromide to the agarose solution (agarose should be completely dissolved)

• 1 ul of EtBr for every 10mL of TAE

Prepare Samples for Sequencing at UW HTGU

1. You'll need two regular PCR plates: one will have your DNA and the other plate the primer that corresponds to the DNA. For example, Primer Plate Well A01 should have the primer that matches the DNA in DNA Plate Well A01. Be sure you know which DNA/Primer is in each well in each plate, as you'll need this information in Step #6!
2. DNA Plate - Requires 10uL of sample in each well. Frequently, it's easiest to decide on a single (i.e. uniform) volume of purified DNA that you'll be able to use from each sample. If less than 10uL, add H2O to each well to make up the difference. Then add your volume of DNA to individual wells.
3. Primer Plate - Requires 10uL of primer at 3uM. Add 7uL of H2O to each well and then add 3uL of the necessary primer (10uM). Remember, only one primer per well. If you need to sequence a particular DNA sample from two directions, you will need two wells in both DNA and Primer plates; one for the forward primer and one for the reverse primer.
4. Seal the plate with aluminum foil plate sealing sheets (in the "Standard PCR Plates/Caps" drawer).
5. Label each plate with your name, date and contents (i.e. DNA or Primers).
6. Fill out the Submission Form (found here as an Excel spreadsheet: http://genefish.fish.washington.edu/~srlab/steven/Forms/DNA%20Sequencing%20Submission%2020080924.xls). Make sure to fill out the plate name at the top of the form. This should match the info that you wrote on your DNA plate in Step #5 above. Also, the form cannot have any "weird" characters. Do not use slashes, hyphens or punctuation (including spaces). If you need a "space", use an underscore.
7. Log in to the sequencing service website here: http://uwhtseq.finchlab. com/Finch/ Ask Steven or Sam for the Username and Password. Click the "Submit" button.
8. Click on the "Place Order" link in the upper left.
9. Select a "Primary Service". You'll pick "Purified PCR".
10. Click the "Choose File" button and find the Submission form that you made in Step #6.
11. Click the "Save & Continue" button.
12. If the file is not accepted, you'll need to correct the errors. Go back to your Excel file from Step #6 and make the appropriate changes, remembering to save the file. Then continue at step #10.
13. If the file is accepted, click the "Please complete your purchasing information" link.
14. Change the "Ship to" drop down menu to the only available choice.
15. Select the "Pay with UW Budget Number" button and enter the correct budget number (must be in this format xx-xxxx).
16. Click the "Save & Continue" button.
17. Click the "Submit Order" button.
18. Open the "sequence_log" Google Spreadsheet and create a new sheet for each Submission Form filled out in Step #6 above. Follow the existing naming scheme for the new sheet. Copy and paste column names and wells from previous sequencing runs.
19. Paste the info from your Submission Form(s) in to the appropriate columns in the new sheet(s) in the "sequence_log" doc.
20. Enter the sequencing transaction in to the "roberts_purchasing_log" Google Spreadsheet. Use $160 as the cost.
21. Bring the plates to Sam (or tell him where they are stored) before 3PM and he will deliver them downtown that day. After 3PM and they may not be delivered until the following morning.
22. Sequencing data is generally available for download from the HTGU website (see Step #7 above) within a week. Sam will notify you when it's available.
11/1/2010 SJW

Run a Protein Gel

Operate the Nanodrop

Operate the Opticon

Wash Dishes

See also: Dish Washer Manual

Operating T21 Ultracentrifuge

Needs attention

Operate Autoclave

Biohazardous material: tightly close the top of each bag with autoclave tape and use the autoclaving basket that is sealed at the bottom
Glasswear: cover the top of each piece of glassware with aluminum foil and tape it down with one strip of autoclave tape, use the baskets without sealed bottoms
(leave the caps of the glassware that have screw on lids slightly unscrewed and put autoclave tape over it)

1. Before opening the lid of the autoclave the pressure gauge should be at zero to avoid getting burned from the steam
2. Use protective gloves to open the lid and remove any other items that were previously or recently autoclaved that could still be fairly hot
3. Make sure items being autoclaved are properly sealed
4. Place items being autoclaved into the proper basket
5. Fill the chamber of the autoclave with 2.5 liters of distilled water
6. Gently insert the basket into the autoclave
7. Tightly seal the lid
8. For glassware make sure all three modes are on (sterilize, exhaust and dry) by pressing the MODE button until all three are lit up
For bioharzardous material only select sterilize by pressing the MODE button until it is the only mode lit up
9. Press START
10. When autoclaving is complete dispose of biohazardous materials in the trash and/or return glassware to its proper cabinet/drawer

Decontaminate Lab Items/Surfaces

Nucleic Acids/Proteins - Any items that cannot (or should not) be autoclaved, wipe contaminated surfaces with a 10% bleach solution. All other items may be autoclaved.

Bacteria/Yeast - Any items that cannot (or should not) be autoclaved, wipe contaminated surfaces with a 10% bleach solution OR a >75% ethanol solution. All other items may be autoclaved. Disposable items should be placed in the appropriate container (e.g. biohazard waste bags, sharps container).

Aquaculture

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Algae Production

Long term storage