RbCl method of competent cell preparation

Posted in Uncategorized by

From the olden days. Sometimes it works great, other times it works terrible.

  1. Inoculate 30 ml of SOB broth in a 250-ml flask with bacteria to be transformed from a single colony on a fresh plate.
  2. Incubate overnight at 37°C with moderate agitation.
  3. Add 8 ml of the overnight culture to a 2-liter sidearm flask containing 300 ml of SOB broth. Incubate at 37°C with moderate agitation until an OD550 of approximately 0.3 is achieved.
  4. Collect the culture in six 50 ml sterile polypropylene centrifuge tubes, and chill on ice for 15 minutes.
  5. Pellet the cells by centrifugation at 3000 x g  for 30 minutes at 4°C. Drain the pellets thoroughly.
  6. Add 2.67mls of ice-cold transformation buffer 1 to each of the six tubes.  Resuspend the pellets by mild vortexing.  Pool into a single 50ml tube.
  7. Incubate tubes on ice for 15 minutes.
  8. Pellet the cells as before (3000 x g 30 minutes, 4°C).
  9. Resuspend the pellet with 16 ml of ice-cold transformation buffer 2.
  10. Aliquot and flash-freeze cells in liquid N2 or dry-ice ethanol bath

Buffers

2 M Mg2+ stock (100 ml)

MgCl2   20.3 g
MgSO4   24.7 g

Dissolve reagents in a final volume of 100 ml dH2O. Sterilize by filtration through a 0.45 µm disposable filter. The resulting solution is 2 M with respect to Mg2+.

0.5 M MOPS (100 ml)

Dissolve 10.47 g MOPS in dH2O. Adjust pH to 6.8 with NaOH. Bring to final volume of 100 ml. Sterilize by filtration through a 0.22 µm disposable filter. MOPS buffer should be clear and colorless.

1 M Potassium acetate (100 ml)

Disolve 9.82 g potassium acetate in dH2O. Adjust pH to 7.5 with acetic acid. Bring to final volume of 100 ml. Sterilize by autoclaving.

SOB Broth (1 liter)

Bacto tryptone           20.0 g
Bacto yeast extract       5.0 g
NaCl                      0.6 g
KCl                       0.5 g
MgCl2                    10 mM (see below)
MgSO4                    10 mM (see below)

Note: SOB is identical to SOC, except that it contains no glucose.

  1. Dissolve tryptone, yeast extract, sodium chloride, and potassium chloride in a final volume of 990 ml distilled H2O. Sterilize by autoclaving.
  2. Add 10 ml of 2 M Mg2+ stock after autoclaving and cooling to the SOB broth to make the media 20 mM with respect to magnesium.

Transformation Buffer 1 (500 ml)

RbCl                6.0 g
MnCl2·4H2O          5.0 g
Potassium acetate  15.0 ml (1 M stock, pH 7.5)
CaCl2·2H2O*         0.75 g
Glycerol           75.0 ml

Combine reagents in dH2O. Adjust pH to 5.8 with 0.2 M acetic acid. Bring to final volume of 500 ml with dH2O. Sterilize by filtration through a 0.22 µm disposable filter. Store at 4°C.

Transformation Buffer 2 (500 ml)

MOPS              10.0 ml (0.5 M stock, pH 6.8)
RbCl               0.6 g
CaCl2·2H2O*        5.5 g
Glycerol          75.0 ml
dH2O          to 500.0 ml

Combine reagents in dH2O. Bring to a final volume of 500 ml with dH2O. Sterilize by filtration through a 0.22 µm disposable filter. Store at 4°C.

* If using anhydrous CaCl2 use 0.57 g for Buffer 1, and 4.15 g for Buffer 2.

Legal NIH Fonts

Posted in Funding, NIH, Uncategorized by

Disclaimer: This is NOT legally binding advice. What follows is my interpretation of the latest (as of 2/25/20) NIH rules regarding acceptable fonts. Those rules were found here and here. I used a similar methodology which is easily visualized in the following figure:

Figure

From the NIH:

In most cases, fonts that meet the “no more than 15 characters per linear inch” requirement will also meet the line spacing requirement when “Line Spacing” is set to “Single”. When in doubt, print off a sample and use a ruler to verify you do not have more than 6 lines within an inch.

I tested EVERY Word 2016 font (PC) set to pt. 11 with default letter spacing as per the figure above. The following fonts were identified as “borderline” or “illegal”. I would note that I considered the borderline fonts unacceptable but your mileage may vary. Every single font that was found unacceptable violated the “15 characters per linear inch” rule EXCEPT  Lucida Console, which violated the “6 lines within an inch” rule. Finally, the NIH claims that conversion to a pdf can change spacing as well. I have my doubts but did not test this claim. (Update: see below)

Borderline
Adobe Devanagari
Bernard MT Condensed
Curlz MT
Gigi
Harlow Solid Italic
Lucida Console (6 lines/in)
PMingLiU-ExtB
Script MT Bold
Sitka Banner
TW Cen MT

Illegal 
Agency FB
Arial Narrow
Bahnschrift Condensed
Bahnschrift Light Condensed
Bahnschrift Semibold Condensed
Bahnschrift Semibold SemiCondensed
Bahnschrift SemiCondensed
Bahnschrift Semilight Condensed
Bahnschrift Semilight SemiCondensed
Blackadder ITC
Bodoni MT Condensed
Bodoni MT Poster Condensed
Brush Script MT
Centaur
Chiller
Edwardian Script
Franklin Gothic Demi Condensed
Franklin Gothic Medium Condensed
Freestyle Script
French Script MS
Gabriola
Gill Sans MT Condensed
Gill Sans MT Ext Condensed Bold
Gloucester MT Extra Condensed
Haettenschweiler
Informal Roman
Juice ITC
Kunstler Script
Microsoft Himalaya
Microsoft Uighur
Microsoft Yi Baiti
Mistral
Monotype Corsiva
Niagara Engraved
Niagara Solid
Onyx
Palace Script MT
Parchment
Perpetua
Playbill
Poor Richard
Pristina
Rage Italic
Rockwell Condensed
TW Cen MT Condensed
TW Cen MT Condensed Extra Bold
Vivaldi
Vladimir Script

Note added in proof: paragraph formatting for the above

Untitled-1

Second note added in proof:

Third note added in proof:

Reviewing Grants

Posted in Funding, NIH, Science, Uncategorized by

I thought it might be useful to outline my process for reviewing NIH grants. I am sure there are many different ways people review grants, however, some general rules apply.

  1. I print double-sided (in color) every grant I am assigned. I review the printed copies and make notes on them directly. Yes, I know I am wasting trees, but sorry this method works well for me. It is also useful in study section because I can see my original notes on each grant.
  2. I read from front to back IN ORDER. That means I am reading the summary/abstract first. Don’t blow off the summary; it sets the tone for me for the rest of the grant. The same goes for the biosketch. The same goes for everything in the grant.
  3. Having to open up the pdf to magnify figures/images so I can understand or read them annoys me. If I still can’t read the figures in the magnified pdf, I am really annoyed. Plus you actually wasted space rather than saving it.
  4. I use a highlighter and pen to make notes on the printed copy. I go through the entire grant from front to back in this manner without typing a single word into the review template.
  5. Yes, I read the vertebrate animal/human subjects section, resource sharing plans, and authentication plans.
  6. I start typing the review. For me, the “overall impact” and “approach” sections are the hardest. Often I will save the “approach” for last and fill out all the other parts.
  7. At this point, I have an idea of what my overall impact score will be, and I try to calibrate my comments to this score. I don’t nitpick a 2 grant, and I provide ample constructive criticism on 6+ grants.
  8. If the mechanism has criterion scores, generally the approach score will closely match the overall impact score. I am not unique in this methodology. 
  9. I upload the reviews/scores only AFTER I have scored all the applications. Sometimes I will go back and re-calibrate my scores based on applications deeper in my pile. Some study sections I have sat on require us to rank our applications in our pile (i.e, “this application was ranked second in my pile of ten”).
  10. As instructed by the SRO, I will occasionally recalibrate incongruent scores before the meeting. This is a rare occurrence in that usually the three assigned reviewers are somewhat in agreement.
  11. At the meeting, I remain completely open to recalibrating my score based upon the discussion (as everyone should be).

Locked Out By BitLocker

Posted in Random, Tech by

My daughter got locked out of her laptop by the Microsoft data encryption program BitLocker. Basically, on startup you get a blue screen prompting for the password. I initially thought this was ransomware or some other malicious software. As I set up her computer, I can say definitively BitLocker was never set up on her computer (by us) nor was a passcode ever provided. Apparently this is a common problem which has been around for years, especially on SurfacePro laptops. The Microsoft position is that there is no way BitLocker can be set up without user input (which appears patently false). Some online solutions revolve around changing BIOS settings. On a relatively new Dell Inspiron laptop, a modified version of the instructions provided in this video worked for me.

  1. Click “Skip this drive”
  2. Click “Troubleshoot”
  3. Click “Advanced Options”
  4. Click “UEFI Firmware Settings”
  5. Click “Restart” – You will restart and enter the BIOS for your computer. The BIOS interface is different for every computer. In my case, it looked nothing like in the above video, and doing subsequent steps in the above video did not work. Doing the following DID work:
  6. Click “Restore Settings” button on the bottom of the Dell BIOS screen. Also see here if this setting is not evident.
  7. Click “Restore Factory Defaults”
  8. Save settings and the computer will reboot. In my case, BitLocker prompt screen is now gone and Windows boots normally.

Coincidentally, BitLocker was also in the news today regarding a massive security flaw with certain solid state drives.

 

Gluten And Dairy Free Chicken Rice Casserole

Posted in Food, Random by

Modified from here.
This is even more delicious that the condensed soup version! Celiac and EOE friendly. Number two in what will hopefully be a larger series of recipes for my daughter.

Ingredients:
Medium yellow onion
1.5 cups rice
2.5 cups beef broth
4.5 tbsp gluten free flour
Olive oil
1 cup unsweetened coconut milk
1 package (8 oz) sliced fresh white mushrooms
Boneless chicken breasts trimmed and quartered (about 4)

Prep about 15 minutes; cook time about 1 hour
1. Preheat oven to 350
2. Chop onion into small pieces
3. Cook onion over medium heat in a skillet in a couple tablespoons of olive oil until tan
4. Add 1.5 cups of beef broth and reduce for 5 minutes
5. While broth is reducing, rub some olive oil liberally in a 9×14 (or similar) glass casserole dish.
6. Add 1.5 cups rice and 1.5 cups water to casserole dish, mix
7. Add broth/onion mixture to casserole dish and mix
8. To skillet (no need to clean), add more olive oil and slight brown mushrooms for a minute or two
9. To 1 cup (cold) beef broth, add 4.5 tbsp GF fluor and mix vigorously; add mixture to mushrooms
10. Immediately add 1 cup unsweetened coconut milk and mix thoroughly
11. Reduce until thick (probably only a minute or two); incorporate into rice mixture in casserole dish until homogenous.
12. Quarter chicken breast and lay on top of rice mixture; season with pepper and salt
13. Cover tightly with foil and cook 1 hour at 350
14. Check to make sure chicken is done by cutting through the thickest piece.

Federal Science Funding: A Call To Action

Posted in Funding, NIH, Science, Uncategorized by

The United States House Science Subcommittee on Energy is responsible for EPA research and development programs. As many of you know, it appears that all EPA grants and contracts have been suspended (at least temporarily). This is a very scary development which tells me no federally funded science is safe. The chair of this committee is Randy Weber, who also happens to be my representative in the 14th district of Texas. I just got off the phone with his office. First, the staffer reiterated the commitment of Mr. Weber to basic science research (not sure why she needed to point this out). Then, she used a lot of words to basically say that states should be responsible for environmental regulation, not the federal government. I noted that my district has several of the largest oil refineries in the nation, and they have repeatedly and willingly polluted our water. In fact, my county has three Superfund sites. I noted that the Texas legislature has a long history of catering to the oil and gas industry, and I don’t trust the state to keep the environment in my county safe. She deflected this by saying that Mr. Weber works with the Texas state equivalent of the EPA known as TCEQ to make sure the oil and gas industry does not pollute our district. I don’t believe it.

Now to the really scary part. In a nutshell, all EPA grants and contracts, both future and present, will be subject to federal oversight before funds are distributed. In other words, government officials will be determining if your research is worthy of funding. Imagine the ramifications for people who study HIV/AIDS, drug abuse, and literally hundreds of other examples of research which may in some way be politicized. This is insanity. Fellow scientists, we have to do something. At the VERY least, call your representatives. This is literally an attack on science. However, I feel we need to do a lot more than just talking to a congressional staffer, and I welcome suggestions.

Thoughts on how to become a PI at an academic institution

Posted in Funding, NIH, Science by

 

eLife published a webinar report on how to get an independent position. On Twitter, Dr. Becca noted that one piece of advice was to apply early before you had any papers from your post-doc position.

To which the following was noted by DM

I have sat on multiple faculty recruitment committees, and I personally have interviewed for what I surmise is probably an above average number of positions. The advice I give for my trainees if they are absolutely serious about becoming a PI at a major institution (particularly medical schools) is this:

  1. Do your post-doctoral training, if possible, with someone at the top of their field. Think HHMI, National Academy, etc.
  2. We all complain about chasing Cell, Science, and Nature papers, but this is what gets you interviews. I am serious. I absolutely don’t agree with this requirement, but I can GUARANTEE there is someone on the search committee who expects this.
  3. Point number 2 is inextricably linked to point  number 1.

I am very aware that there are hundreds of people who did not have CNS or famous people on their CV and still got a position. Also, these points are usually only important for getting an interview. The qualities required for a successful interview are completely different. Anyhow, DM is right, the rules are different depending on who/where you worked. Furthermore, it also greatly influences your trajectory downstream of getting that first position.

Postscript from the Twits:

Why You Can’t Use A Google Scholar Link In Your Biosketch

Posted in Uncategorized by

I submitted a request for clarification to grantsinfo@nih.gov for the following policy:

Indicate that a URL for a publication list is optional and, if provided, must be to a government website (.gov) like My Bibliography.

This was in response to recently updated instructions for preparing the biosketch and some discussion on Twitter initiated by @crytogenomicon.

https://twitter.com/cryptogenomicon/status/730412499120566272?lang=en

After about a week, I got the following reply:

With broader rules for including links we could not protect the anonymity of our reviewers.

It took me a few minutes to process this result, and then it hit me. I could link, for example, to the publication list on my personal lab web page. I use Google Analytics, and thus I can see where traffic to my site originates from. It would be very simple to figure out who reviewed my grant in this scenario. Turns out @thatdnaguy was right!

https://twitter.com/thatdnaguy/status/731447635819646976?lang=en

Reproducibility

Posted in Career, Science by

When I was a grad student, I modified an existing technique to measure the activity of an ion channel that resides on intracellular membranes. The major advance was that I extended the so-called “nuclear patch-clamp technique” from Xenopus nuclei to mammalian cells*. For several reasons that are unimportant, this was of fairly high interest to the field and immediately people tried to replicate my findings. It turns out that like many things in science, there are subtle aspects to the protocol that greatly increase the probability of success which do not translate well to words on a written protocol. I literally traveled to several labs around the country to show them with my own hands how to do this type of recording. This is now routine in the field, with a couple groups who are exceptionally good at this technique getting a lot of collaboration requests. The point is that if you can’t reproduce something, contact the group who published the work and they will likely help you in any way you can. I have certainly benefitted from this, as our most recent paper** required many email/phone calls to a couple of other groups who were much more competent in the assays we were trying to perform.  One last thing, the literature is inherently self-correcting. The notion that you cannot publish contradictory or negative results is a fallacy***.

*http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1301497/

**http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586849/

***http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572306/

 

Study Sections Are Not Evil

Posted in Career, Funding, NIH by

Science twitter is a great place to meet new scientists, find new collaborators, and learn a lot about our sometimes maddening profession. It allows us to frankly discuss (among other things) career development, workplace issues, and publishing models. Sometimes I fear us older* scientists whine enough to discourage some of our younger followers from pursuing a career in science, and in particular academic science. This likely comes from our struggles to obtain grant funding from the NIH, NSF, and other agencies which have seen devastating reductions in budgets over the past decade or so. With uncertainty and failure comes blame, and study sections are easy pickings to vent our frustration. I just got off study section yesterday. All of us on the panel are in the same horrible funding environment, and we realize that the people applying are our peers or future peers. I would like to believe that yesterday we all followed the mantra “review unto others as you would have them review unto you”. The meeting was very useful to come to a consensus on the not insignificant percentage of apps in which we had a difference of opinion. In retrospect, I feel we were fair, balanced, and did the best job possible. I have always felt this way after study section, no matter the agency. So don’t blame the study section. For the most part, we are doing the best we can do.

*FTR, I do not consider myself old. I can’t believe I have been a PI over a decade now.