When I started this blog in 2012, I did it to make sense of my thoughts about starting my first faculty position as an Assistant Professor and to share with a few friends. Never ever I would have thought 1) that I would still be doing this seven years later, 2) that the blog would have more than 500,000 total views, and 3) that I would have become part of a community of science bloggers and Twitter users who supported me scientifically and personally for this long. On a fateful day in 2014 when Melissa @biochembelle tweeted about my posts and introduced me to the scientific community on social media everything changed. I made lots of pocket friends, many of whom became real-life friends, and I was introduced to an extended support network full of information and new ideas, often providing respite from the lonely life of starting a lab on Twitter and via the @NewPI_slack. I met colleagues who were a sounding board for grant ideas and who helped with my career progression and transition (you know who you are). I found Peg AtKisson @iGrrrl who was hired as a grant consultant and was instrumental in helping me get 2 R01s. I watched science Twitter dynamics oscillate between chaotic good and chaotic evil sometimes, and tried my best to tip the scales towards good. I learned a lot about diversity and was inspired to build a more inclusive environment wherever I go. So first of all, thank you!
With the general decline of blogging and the trend toward shorter and more immediate forms of sharing information, I was wondering if this would still be useful, but when I asked 94% of respondents said they would still want a blog. And checking the stats I realized that this site still gets 3-4,000 hits a month despite me not having posted in 10 months...so I will keep going. Since I'm not a new PI any longer and readers asked to focus on mid-career faculty issues, I decided to start a new blog My Mid-career Academic Life. I will try and cross-reference the two blogs at the beginning to get clicks and get indexed and all the necessary internet things, but I didn't want to change the identity of The New PI blog and I prefer to move on. I've been meaning to unpseud for a while, but today is not the day and I promise it will be soon (-ish).
In following @drugmonkeyblog's tradition to list the posts of the month at the end of each year, I decided to link 12 posts to close this blog: the 6 most read and 6 favorite ones which may not have had as much attention. Here we go.
Greatest hits:
1) Submitting your R00 proposal to transfer your K99 to your new job: a survival guide - 12,547 hits as of today. I'm so happy this has been helpful for so many of you!
2) Interviewing for a postdoc: questions you should ask - 11,176 hits. This was for the trainees.
3) A compilation of K99 and R00 advice - 10,890 hits. This links to a lot of my other K99/R00 posts and other blogs/articles that could be useful in the process. I may skip some other popular K99 posts because they're all included here.
4) Where do you find grant money for your lab? - 5,939 hits. This is old and has not been updated in a while, but it's still a good starting list.
5) Tales of postdocs past: what did I learn? - 5,638 hits. Cultivating or identifying resilience in trainees is something faculty discusses ad nauseam and this was one of the first times I articulated some thoughts on finding the right lab peeps.
6) Will I have jumped the glass cliff in 5 years? - 5,554 hits. Looks like the answer to that question is NO, but this was definitely one of my most emotional posts and it struck a chord with many people.
My personal faves:
1) The New PI hits the 6 month slump: how do you keep proactive? - The first 6 months of a faculty position are so hard! Whenever I bump into friends at conferences who have just started a lab, I think of this post and I just want to hug them.
2) Musings about work ethics and an unstructured schedule - I still stand by this one.
3) Is resilience the name of the game in academia? - Yes, it is. Happy to report that yours truly, Doc Becca and my nameless friend in this post are all Associate professors and NIH funded.
4) The beginning of the R01 twin strategy - This sounded crazy and controversial and it kind of worked...not as I intended because they ended up being Irish twins. But yay, twins!
5) In the belly of the beast. NIH Early Career Reviewer - Part 2 of a two-part post on the NIH ECR program. What I learned about NIH review in my first experience is still helping me now that I've been Reviewer 1.
6) Deciding which university is the right fit for you - A more recent and mature post on how to decide which environment you want to be in and how to find it.
So long, and thank you for all the fish! 🐬🐬
Showing posts with label Lab stuff. Show all posts
Showing posts with label Lab stuff. Show all posts
Thursday, December 26, 2019
Monday, May 19, 2014
Freezers and fridges for your lab
The pricing of research items does not make sense to me. When I tried to buy a drafting stool for a high bench from Overstock.com for $110, I was told that insurance only covers chairs bought from VWR or Fisher and had to spend $244 for a VWR High Bench chair, luckily deeply discounted from a list price of $674.
As the lab is growing we started to fill the hand-me-down freezers and fridges I had collected from other investigators, and I had to figure out what to do. I wanted a nice and spacious 2 door deli case fridge. I didn't need a fancy chromatography fridge such as the one listed from VWR for $9,695.69 (Symphony Chromatography Refrigerators), but even the basic 49 cu. ft. 2-door fridge was not much cheaper at $6,899. With a good quote, I could have probably knocked 2-3K off the list price, but even if my institution has restrictions on chairs, it does not have restrictions on vendors for appliances. So after multiple searches on Webstaurantstore.com and KaTom Restaurant Supply, in comes a great 45 cu. ft. 2-door beverage merchandiser from True Refrigeration for around $2,300. I paid an extra $400 for white glove delivery so that it wouldn't get stuck in the loading dock and our fridge space is now tripled. A neighbor has had the same fridge happily for 10 years...and even if it breaks in 5 years, it's still 1/3 of the scientific distributor's price. This is $3-5,000 in your equipment budget that now you can use for something else.
Same goes for small undercounter freezers and fridges. In my old institution every bench came with one undercounter for free I believe, but here I just need a couple for specific applications that require separate freezer space. Again the VWR 5 cu. ft. general purpose freezer is listed at $1,639. From Best Buy you can get a 4.2 cu. ft. upright freezer for less than $300.
Hope this helps.
 |
| Snowflakes at the electron microscope |
Same goes for small undercounter freezers and fridges. In my old institution every bench came with one undercounter for free I believe, but here I just need a couple for specific applications that require separate freezer space. Again the VWR 5 cu. ft. general purpose freezer is listed at $1,639. From Best Buy you can get a 4.2 cu. ft. upright freezer for less than $300.
Hope this helps.
Wednesday, May 7, 2014
Setting up your lab website
Your website is your labs' ambassador to the outside world. Prospective students and job applicants will check it out before applying, so will collaborators or potential donors. I have been talking to a lot of other new PIs discussing website options and set up my site right when I opened the lab. In our hyper-connected world not having a website is almost like being invisible.
I made some choices, which may not necessarily be the best ones, but I thought I'd share the process.
Independent vs. institutional. In some cases this may not be a choice at all. Some school will be very protective of their brand and will not allow their logo to be used in your personal lab website. In this situation, they may offer to host your site, but you will have very limited personalization options and will end up with a site which will look like your institution's site. As scientists we tend to want to express our individuality and to attract the best applicants, we also want to project the lab personality on the site. Is your brand distinct from the one of your institution? How much are you willing to pay for your brand?
Homemade vs. pre-made vs. custom designed. The choice on the design of your independent site will come down to 1) how good you are with HTML and 2) how much you care or want to spend. If you or a student of yours are good a web design, you can make your site whatever you want, but have to take the time to generate the look. If you are willing to spend some money to use a hosting service with pre-designed templates, you can pick something you like and populate the template much faster. Then, there is always the option of having a professional get a personalized template ready for you to populate.
Fancy vs. simple. The choice of which design to use is not an easy one. In general my feeling is that lab websites have to be relatively simple without crazy graphics and busy columns, but a site with more bells and whistles may be more attractive to a younger audience. At the end your site must reflect the image of the lab.
A word about hosting. There are many different hosting options that could be available to you. If your university does not care about branding, they may be willing to host your personal lab site for you with your design. If you want to have an outside provider and what to have a pre-designed site there are multiple hosting sites ranging from $0.50 to $5 a month for basic service. It's not easy to determine which hosting service would work best and at the end it may just come down to cost, template availability and ease of update. Some comparisons can be found here and here. I am using iPage, which as far as I can tell has been very good, templates are good looking and customizable and updates are very easy. The structure of the pages could probably be more flexible, but it works well for a basic lab website.
I made some choices, which may not necessarily be the best ones, but I thought I'd share the process.
Independent vs. institutional. In some cases this may not be a choice at all. Some school will be very protective of their brand and will not allow their logo to be used in your personal lab website. In this situation, they may offer to host your site, but you will have very limited personalization options and will end up with a site which will look like your institution's site. As scientists we tend to want to express our individuality and to attract the best applicants, we also want to project the lab personality on the site. Is your brand distinct from the one of your institution? How much are you willing to pay for your brand?
Homemade vs. pre-made vs. custom designed. The choice on the design of your independent site will come down to 1) how good you are with HTML and 2) how much you care or want to spend. If you or a student of yours are good a web design, you can make your site whatever you want, but have to take the time to generate the look. If you are willing to spend some money to use a hosting service with pre-designed templates, you can pick something you like and populate the template much faster. Then, there is always the option of having a professional get a personalized template ready for you to populate.
Fancy vs. simple. The choice of which design to use is not an easy one. In general my feeling is that lab websites have to be relatively simple without crazy graphics and busy columns, but a site with more bells and whistles may be more attractive to a younger audience. At the end your site must reflect the image of the lab.
A word about hosting. There are many different hosting options that could be available to you. If your university does not care about branding, they may be willing to host your personal lab site for you with your design. If you want to have an outside provider and what to have a pre-designed site there are multiple hosting sites ranging from $0.50 to $5 a month for basic service. It's not easy to determine which hosting service would work best and at the end it may just come down to cost, template availability and ease of update. Some comparisons can be found here and here. I am using iPage, which as far as I can tell has been very good, templates are good looking and customizable and updates are very easy. The structure of the pages could probably be more flexible, but it works well for a basic lab website.
Sunday, November 24, 2013
Biotium GelRed and GelGreen to replace ethidium bromide
We would like to run agarose gels without ethidium bromide to avoid the need of dealing with toxic waste and to reduce the use of dangerous chemicals in the lab. We talked to quite a few vendors about this and started trying things out.
We started with AMRESCO EZ-Vision dyes which are SYBR Green based and were not very happy with them. Staining was very weak and we could not reliably image the gels on our existing UV image acquisition system without a SYBR filter. We had no intention of buying a new image acquisition system just for this so we kept looking. 500uL $89.52 from VWR
We tried Promega Diamond Nucleic Acid dye was also pretty weak and it is not an in-gel stain, so requires additional time for post-staining. We decided to keep looking. 500ul $70.00 from Promega
Finally, we started having good results with Biotium GelRed. GelRed is very sensitive and looks orange/red on the UV transilluminator like EtBr. GelGreen was not quite as bright, so we focused on GelRed, but optimization was needed. We had two primary issues: 1) bands which are very close to each other appear like they are fused together and the molecular weight marker was just a smear, 2) bands sometimes ran at a different weight than expected.
Since one of our genotyping PCRs needs to distinguish between a 400 and 480bp band it was very important to get good separation. Biotium recommended to reduce the amount of dye in the gel or to try post-staining because both the separation issue and the run length issue could be due to the larger size of the Biotium dye which can impair DNA motility. We lowered the amount of in-gel stain from 10,000X to 40,000X and it still worked pretty well, but a range of 20-30,000X may work best (around 2ul for a 50mL gel). Another way to improve separation is to post-stain if necessary and post-staining is relatively quick (15-20 mins) and does not require destaining. In addition the post-staining dye can be reused saving some extra money. Because of the size of the dye, agarose concentration is also critical and concentrations above 1% are not recommended. 500ul $104.42 from VWR
All in all for regular genotyping PCR which is most of what we do the GelRed works very well and while it's somewhat more expensive than the other dyes, it is a substantial difference in sensitivity. In the end the decision comes to whether you want to have an EtBr free gel bench and how much money you are going to spend for the more expensive dye.
5g of EtBr ($64.20) will make 1L of 10,000X solution (final concentration 5mg/mL), while 1L of GelRed at 30,000X would cost $35,000. Yet, 1L of stock will make 100,000 100mL gels: if you run 10 100mL gels a week or around 500 gels a year, you'll spend $0.32 in EtBr or $417.68 in GelRed. I would be interested in finding out how much you spend yearly for EtBr disposal.
We started with AMRESCO EZ-Vision dyes which are SYBR Green based and were not very happy with them. Staining was very weak and we could not reliably image the gels on our existing UV image acquisition system without a SYBR filter. We had no intention of buying a new image acquisition system just for this so we kept looking. 500uL $89.52 from VWR
We tried Promega Diamond Nucleic Acid dye was also pretty weak and it is not an in-gel stain, so requires additional time for post-staining. We decided to keep looking. 500ul $70.00 from Promega
Finally, we started having good results with Biotium GelRed. GelRed is very sensitive and looks orange/red on the UV transilluminator like EtBr. GelGreen was not quite as bright, so we focused on GelRed, but optimization was needed. We had two primary issues: 1) bands which are very close to each other appear like they are fused together and the molecular weight marker was just a smear, 2) bands sometimes ran at a different weight than expected.
Since one of our genotyping PCRs needs to distinguish between a 400 and 480bp band it was very important to get good separation. Biotium recommended to reduce the amount of dye in the gel or to try post-staining because both the separation issue and the run length issue could be due to the larger size of the Biotium dye which can impair DNA motility. We lowered the amount of in-gel stain from 10,000X to 40,000X and it still worked pretty well, but a range of 20-30,000X may work best (around 2ul for a 50mL gel). Another way to improve separation is to post-stain if necessary and post-staining is relatively quick (15-20 mins) and does not require destaining. In addition the post-staining dye can be reused saving some extra money. Because of the size of the dye, agarose concentration is also critical and concentrations above 1% are not recommended. 500ul $104.42 from VWR
All in all for regular genotyping PCR which is most of what we do the GelRed works very well and while it's somewhat more expensive than the other dyes, it is a substantial difference in sensitivity. In the end the decision comes to whether you want to have an EtBr free gel bench and how much money you are going to spend for the more expensive dye.
5g of EtBr ($64.20) will make 1L of 10,000X solution (final concentration 5mg/mL), while 1L of GelRed at 30,000X would cost $35,000. Yet, 1L of stock will make 100,000 100mL gels: if you run 10 100mL gels a week or around 500 gels a year, you'll spend $0.32 in EtBr or $417.68 in GelRed. I would be interested in finding out how much you spend yearly for EtBr disposal.
Saturday, August 17, 2013
Why we like Quartzy for ordering, inventory and reagent organization
Starting a lab means that you get to establish culture and workflow, which I have previously discussed here, and one of the main problems I have encountered in the past is the lack of a proper inventory and common reagents constantly running out stopping experiments dead in their tracks. If you are in a large lab, you can obviate to this problem by having your own private stock of everything that is vital to your research, but I am starting a new lab which is small, and I want sharing and good labmanship to be a strong feature of my group.
I was on LabLife for a few years and I was never really into it because nobody else in the lab used it, and now that it has transitioned from a free version to the paid version, LabGuru, paying for it is out of the question. It costs $10/month per person and I am not willing to pay the much for every postdoc, student and undergrad to have access to inventory.
We have a Dropbox folder where I keep all the protocols and we share data, and I'm looking into getting server space for more data storage. But now that we ORDER A LOT to get the lab set-up, I also need a place where every order, quote and part number is recorded and where we can write down where everything is. My postdoc lab has recently established a Google spreadsheet for orders with a template that everyone can access to enter what they need. Everyone can then go check when the order has been placed and whether it has been delivered. This is easy and free, but again it's a one trick pony.
So, after seeing all the little fliers for Quartzy, we decided to give it a try and absolutely love it!!
Starting with the Orders tab anyone in the lab can put in an order and when the system is integrated with a particular vendor's catalog the order is auto-populated with all kinds of useful information. Then the order is submitted to the approver (i.e. me or a lab manager) who orders stuff and marks it as Ordered. I think in some institutions you can order directly via Quartzy, we are not allowed to do that and mostly have to use iBuy. When stuff comes whoever unpacks it marks it Delivered and has the option to indicate where the items are now located AND automatically everything that was delivered ends up in Inventory. Yay! Handy searches and item categories (Equipment, Antibody, etc) make finding stuff in the inventory very easy and the view for each category is fully customizable so that the most important info is easily available, like serial number for equipment vs epitope for antibodies. There is a tab for Protocols and one for Facilities and while I don't use the Protocols because ours are in the Dropbox, I already set up the sign-up sheets for our large equipment and I'm excited to use the scheduling function.
While entering everything at the beginning is very time consuming, I'm hoping this will pay back in the future. We recorded all the special prices and quotes we received as a new lab and if something is running low the person noticing this will just have to click Reorder. If someone is wondering where stuff is, they just have to look it up in the Inventory. In addition, if you have many freezer boxes of samples and reagents and you record the exact location of every tube, you can generate a box map and a list, which saved us from having to get FileMaker or equivalent database software.
Finally, the customer service is absolutely wonderful. The chat box on the right allows you to IM a customer service rep on the spot. I had an issue with the ordering layout because there was no easy way to keep track of the ordered and delivered dates. While the rep could not help me right then and there, she told me they were working on it and two days later the layout changed to give you a much better overview of what is going on. I'm really excited to see how Quartzy changes in the future and I would urge everyone who uses the site to give lots of feedback so that it can become even more user friendly. Now, I just hope they don't go the LabLife way and charge an arm and a leg for it.....
I was on LabLife for a few years and I was never really into it because nobody else in the lab used it, and now that it has transitioned from a free version to the paid version, LabGuru, paying for it is out of the question. It costs $10/month per person and I am not willing to pay the much for every postdoc, student and undergrad to have access to inventory.
We have a Dropbox folder where I keep all the protocols and we share data, and I'm looking into getting server space for more data storage. But now that we ORDER A LOT to get the lab set-up, I also need a place where every order, quote and part number is recorded and where we can write down where everything is. My postdoc lab has recently established a Google spreadsheet for orders with a template that everyone can access to enter what they need. Everyone can then go check when the order has been placed and whether it has been delivered. This is easy and free, but again it's a one trick pony.
So, after seeing all the little fliers for Quartzy, we decided to give it a try and absolutely love it!! Starting with the Orders tab anyone in the lab can put in an order and when the system is integrated with a particular vendor's catalog the order is auto-populated with all kinds of useful information. Then the order is submitted to the approver (i.e. me or a lab manager) who orders stuff and marks it as Ordered. I think in some institutions you can order directly via Quartzy, we are not allowed to do that and mostly have to use iBuy. When stuff comes whoever unpacks it marks it Delivered and has the option to indicate where the items are now located AND automatically everything that was delivered ends up in Inventory. Yay! Handy searches and item categories (Equipment, Antibody, etc) make finding stuff in the inventory very easy and the view for each category is fully customizable so that the most important info is easily available, like serial number for equipment vs epitope for antibodies. There is a tab for Protocols and one for Facilities and while I don't use the Protocols because ours are in the Dropbox, I already set up the sign-up sheets for our large equipment and I'm excited to use the scheduling function.
While entering everything at the beginning is very time consuming, I'm hoping this will pay back in the future. We recorded all the special prices and quotes we received as a new lab and if something is running low the person noticing this will just have to click Reorder. If someone is wondering where stuff is, they just have to look it up in the Inventory. In addition, if you have many freezer boxes of samples and reagents and you record the exact location of every tube, you can generate a box map and a list, which saved us from having to get FileMaker or equivalent database software.
Finally, the customer service is absolutely wonderful. The chat box on the right allows you to IM a customer service rep on the spot. I had an issue with the ordering layout because there was no easy way to keep track of the ordered and delivered dates. While the rep could not help me right then and there, she told me they were working on it and two days later the layout changed to give you a much better overview of what is going on. I'm really excited to see how Quartzy changes in the future and I would urge everyone who uses the site to give lots of feedback so that it can become even more user friendly. Now, I just hope they don't go the LabLife way and charge an arm and a leg for it.....
Tuesday, June 18, 2013
Never pay retail....in the lab
I am an avid shopper and I love nice things, but as a grad student and postdoc I didn't have much money, so I've become an expert on where to find the best bargain and how to wait for the things I wanted until I could get the most favorable price. I doubt it's safe to buy lab supplies on eBay (though there are quite a few things available), but in a few months of running a lab I discovered that the "never pay retail" rule is also true for labs.
Every new principal investigator (PI) knows about the new lab discounts you can get from multiple companies, but what I didn't know is that you should never be satisfied with those and that you can get discounts all the time....for almost everything. You just need to ask, and better you need to come armed with quotes from multiple companies and from pricing that was extended to other PIs. I have quite a few friends that have started labs in the past couple of years plus multiple people at my new institution, so I've been collecting quotes and deals from everyone. Then I can go to the different vendors and get quotes and pit them against each other and against the best prices I have heard of. This is time consuming and it is sometimes uncomfortable since you may develop a rapport with certain vendors and you may be genuinely torn between different products, but the dance is worth your time. If a company offers 30% off and you like another product better, you should ask the competing company to extend the same discount. Everything is ridiculously expensive and your start-up money is very precious.
Some things I've learned:
- If you are in the market for a good microscope and have found one you like, ask whether demos are available. Demo microscopes are just lightly used and you can get a better price for what you want or some free bells and whistles which could become handy.
- Always see if you can get free reagents/consumables with your equipment purchase and if you get a $1000 ask for a quote for what you need with the new lab discount and get even more stuff for free.
- Find out if your institution allows you to purchase your fridges and freezer for a regular stores. Best Buy will have freezer for a fraction of the price of big distributors like VWR or Fisher and you can find deli case fridges from restaurant supply stores at half to a third of the price. Google "2 door beverage merchandisers" and you'll see.
- The price of chemicals may vary wildly and one friend recommended to always double check since she level of purity does not necessarily make something more expensive. Companies will make the most requested item the cheapest, so you may be able to get a better purity for a smaller price tag.
- Always always ask for a better discount....and always ask around for what your friends and colleagues are doing. What was the norm in your old lab could be done with alternative approaches that are much cheaper and there may be other things you want to splurge on.
Every little bit counts.
Saturday, April 27, 2013
Qiagen Finder 45-pathway Cignal array: the good, the bad and the ugly
I am still not sure whether this is going to be a good review or a bad review. It may shake out one way or another as I write it.
We do a lot of signaling as we work on a intracellular signaling scaffold which functions in a lot of different pathways, so I always pay attention to new email from SABiosciences/Qiagen on their new PCR and luciferase arrays. Upon reading about it the Cignal Finder 45-pathway array seemed to provide an awesome way to apply very sensitive luciferase assays to test 45 pathways in a single shot.
It is a 96 well plate with 45 reporters in duplicates and 3 positive and negative controls that you can use to test you drug, siRNA, over expression plasmid on your cell line of choice. The protocol is simple and straightforward: the DNA is in the wells, you add the cells and transfection reagent and the next day you test for luciferase and renilla activity. At $1,600 for a 10 plate pack, plus dual-luciferase assay reagents, it can run around $200-300 per plate, which in triplicates for 2 conditions can be almost $2,000 per experiment. But screening 45-pathways at once could be totally worth it and SABiosciences always run some kind of deal on the plates (and you can use a fraction of the luciferase reagent recommended in the Promega protocol).
We know our protein of interest would really strongly activate NF-kappaB and we have done it with a different reporter, so that was our positive control. The first plate was amazing! There were very clear difference between reporters, the duplicates looked very consistent and a whole bunch of interesting pathways lit up in addition to NF-kappaB in our experimental condition. The dynamic range of response was incredible with some reporters with intensity in the 2,000,000 intensity units and some in the 2,000. And so we embarked in a huge study with over expression and siRNA...and ordered another 10-pack. Qiagen provided us with a handy Excel file for analysis where we just needed to plug the results of luciferase and renilla for each plate and everything would be calculated and plotted for us.
I was intrigued by some of the results and started paying a lot of attention to the intensity measurements, and that is when things started getting ugly. We had noticed that some of the duplicates were asymmetric in intensity when a low intensity reporter was next to a very high intensity one. I had had similar problems in the past with white plates in our luminometer, where we noticed spillover of fluorescence in neighboring wells. Some people say it's the fluorescence passing trough the white walls, others that the detector is far from the plate and the fluorescence spills over from above like a halo. Also instead of looking at the results in the Qiagen spreadsheet where they are in linear format, I analyzed everything in plate format, where I could see which well was above, below, left and right of any particular reporter. That is when half of our hits became uninterpretable because they could simply be due to spillover from the strong hit next door. Qiagen recommended to half the amount of lysate, but that didn't help much as the results were basically identical. So we were stuck with the option of spending $500 per troubleshooting attempt or find some other solution....
They said it must be our luminometer, which is a possibility, but that's the machine we have in the department and their product should work even for older machines. They gave us 2 plates for free for testing, which was nice, but still leaves for $500 per troubleshooting for each one of the other variables (machine, reagents, lysate amount). Right now I am faced with a conundrum: I have some data I believe from the array and some data I do not believe, which I think is due to spillover. I cannot publish the whole array because I would have to explain it is a problematic product and I have requested for a sample of the 6-8 reporters that are iffy, but I was told I had to buy them individually, which I am not inclined to do at this time, since I do not want to waste my grant money to troubleshoot their product for them.
At the same time, their reporters work beautifully and the data we have is very robust, though for us it was more a 30-pathway array than a 45-pathway because of the issues.....I am open to suggestions from other people who may have tried the product and who may have other ideas. Also, I am not entirely satisfied with the way they recommend to analyze the reporters and I'd love input on that also.
We do a lot of signaling as we work on a intracellular signaling scaffold which functions in a lot of different pathways, so I always pay attention to new email from SABiosciences/Qiagen on their new PCR and luciferase arrays. Upon reading about it the Cignal Finder 45-pathway array seemed to provide an awesome way to apply very sensitive luciferase assays to test 45 pathways in a single shot.
It is a 96 well plate with 45 reporters in duplicates and 3 positive and negative controls that you can use to test you drug, siRNA, over expression plasmid on your cell line of choice. The protocol is simple and straightforward: the DNA is in the wells, you add the cells and transfection reagent and the next day you test for luciferase and renilla activity. At $1,600 for a 10 plate pack, plus dual-luciferase assay reagents, it can run around $200-300 per plate, which in triplicates for 2 conditions can be almost $2,000 per experiment. But screening 45-pathways at once could be totally worth it and SABiosciences always run some kind of deal on the plates (and you can use a fraction of the luciferase reagent recommended in the Promega protocol).
We know our protein of interest would really strongly activate NF-kappaB and we have done it with a different reporter, so that was our positive control. The first plate was amazing! There were very clear difference between reporters, the duplicates looked very consistent and a whole bunch of interesting pathways lit up in addition to NF-kappaB in our experimental condition. The dynamic range of response was incredible with some reporters with intensity in the 2,000,000 intensity units and some in the 2,000. And so we embarked in a huge study with over expression and siRNA...and ordered another 10-pack. Qiagen provided us with a handy Excel file for analysis where we just needed to plug the results of luciferase and renilla for each plate and everything would be calculated and plotted for us.
I was intrigued by some of the results and started paying a lot of attention to the intensity measurements, and that is when things started getting ugly. We had noticed that some of the duplicates were asymmetric in intensity when a low intensity reporter was next to a very high intensity one. I had had similar problems in the past with white plates in our luminometer, where we noticed spillover of fluorescence in neighboring wells. Some people say it's the fluorescence passing trough the white walls, others that the detector is far from the plate and the fluorescence spills over from above like a halo. Also instead of looking at the results in the Qiagen spreadsheet where they are in linear format, I analyzed everything in plate format, where I could see which well was above, below, left and right of any particular reporter. That is when half of our hits became uninterpretable because they could simply be due to spillover from the strong hit next door. Qiagen recommended to half the amount of lysate, but that didn't help much as the results were basically identical. So we were stuck with the option of spending $500 per troubleshooting attempt or find some other solution....
They said it must be our luminometer, which is a possibility, but that's the machine we have in the department and their product should work even for older machines. They gave us 2 plates for free for testing, which was nice, but still leaves for $500 per troubleshooting for each one of the other variables (machine, reagents, lysate amount). Right now I am faced with a conundrum: I have some data I believe from the array and some data I do not believe, which I think is due to spillover. I cannot publish the whole array because I would have to explain it is a problematic product and I have requested for a sample of the 6-8 reporters that are iffy, but I was told I had to buy them individually, which I am not inclined to do at this time, since I do not want to waste my grant money to troubleshoot their product for them.
At the same time, their reporters work beautifully and the data we have is very robust, though for us it was more a 30-pathway array than a 45-pathway because of the issues.....I am open to suggestions from other people who may have tried the product and who may have other ideas. Also, I am not entirely satisfied with the way they recommend to analyze the reporters and I'd love input on that also.
Thursday, February 14, 2013
A valentine to the Applied Biosystems Veriti® Thermal Cycler
You know how as a child you see things adults have, and as a grown-up you buy the same things so that you feel like an adult? PCR machines are it for me: a lab is not a lab without one. We demoed a Veriti® last Fall and really liked it, then got an offer that we couldn't refuse with a bonus on the 2-for-1 end-of-year special, which made them really affordable.
What is so special? Well, first of all, as you can find in a handy video here, the Veriti® is really easy to use, you plug it in and 5 minutes later you are running your samples. The interface is completely intuitive and the touchscreen is very responsive. THEN, with the VeriFlex™ block you can use 6 separate temperatures for two 8-well strips at a time. This means not only that you can run a precise gradient to test your conditions, but also that you can run multiple reactions at different melting temperatures as long as the extension is the same. We tried both these things with our very difficult genotyping reaction and I almost wanted to cry it was so beautiful. We ran both the wild-type and knock-out reactions on the same block with a 50 and a 58 degree melting temp at each end and everything worked. Since some of our more complex breeding needs up to 4 different PCRs to capture all the alleles, we would be able to run everything at the same time on 1 or 2 machines. We have not tested neighboring strips yet for consistency and interference. They claim you can do completely different temperatures, but we just built a consecutive step gradient and used opposite ends so far.
Finally, apparently you can plug the machine into the Internet and there is software available so that you can run it remotely or have it send you an email when the reaction is done. This is a little creepy. I'm not sure I want a thermocycler to have that much control over my life, but it could be a handy feature if you are doing a lot of things and tend to forget, or if you're going out for dinner and want to know when to get back to the lab. Though I think the 4-10 degree hold should be sufficient for that...DNA is pretty stable....
Remember to look at the Lab-things we like page for more products and reviews.
Saturday, January 26, 2013
Millipore SNAP i.d. protein detection system
SNAP i.d. is a vacuum-driven apparatus to block, stain and wash your Western blots. It promises to shorten your time to as little as 30 minutes from the moment you finish your transfer to the moment you are ready for detection. It's definitely a significant improvement from almost 24 hours.
The concept is simple you put your membrane in a special cassette which allows even distribution of liquids, you first put the block on top and the vacuum sucks it through, then you continue with the primary antibody, etc. Blocking takes the time for 30ml of solution to flow through the system and antibody stainings which are done in 3 mL of liquid are 15 minutes. Washes are immediate. There is even a little plastic collector you can put under the cassette to recover your antibody, if you wish.
Does it work? Yes. We were really on the fence on whether we wanted to buy it. At $1,500 it is not cheap, but would it provide such huge time savings to be worth the price? If you are a biochemistry lab working on a finite set of antibodies and repeating the same assays over and over again, absolutely. We calculated the cost per run and since the antibody itself is the most expensive fraction of the cost, the $2 per cassette is quickly compensated by having to use less total antibody (though they recommend 3X concentration). At the end the cost per run is the same with traditional methods than with the SNAP i.d. In addition, cassettes can be reused a few times for the same antibody which would make it even cheaper.
The problem is optimization. Every antibody must be optimized as far as concentration and incubation time. Reviews on the internet (here and here) indicate that sometimes you cannot skip the usual overnight primary incubation because the fast antibody absorption just doesn't work and that you have to play with the antibody concentration because of reduced sensitivity. One possibility we considered is to use it just for secondaries, which would shorten the washes-secondary-washes portion to 20 minutes instead of 3 hours.
The concept is simple you put your membrane in a special cassette which allows even distribution of liquids, you first put the block on top and the vacuum sucks it through, then you continue with the primary antibody, etc. Blocking takes the time for 30ml of solution to flow through the system and antibody stainings which are done in 3 mL of liquid are 15 minutes. Washes are immediate. There is even a little plastic collector you can put under the cassette to recover your antibody, if you wish.
Does it work? Yes. We were really on the fence on whether we wanted to buy it. At $1,500 it is not cheap, but would it provide such huge time savings to be worth the price? If you are a biochemistry lab working on a finite set of antibodies and repeating the same assays over and over again, absolutely. We calculated the cost per run and since the antibody itself is the most expensive fraction of the cost, the $2 per cassette is quickly compensated by having to use less total antibody (though they recommend 3X concentration). At the end the cost per run is the same with traditional methods than with the SNAP i.d. In addition, cassettes can be reused a few times for the same antibody which would make it even cheaper.
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| Scilogex Analog Tube Roller |
As a new lab, I decided to pass for the moment because the upfront cost and the cost of consumables does not justify the expense. Instead we bought a Scilogex tube roller, which cost around $250, so that we could incubate our primaries in rolling Falcon tubes instead of shaking plastic cassettes cutting down the antibody solution to 2-3mL at the usual concentration, which saves us from using the 3X concentration required from the SNAP i.d. And the thing about the tube roller is that you can use it for a lot of other things. One day when I am rich, I'll buy a SNAP i.d.
Remember to look at the Lab-things we like page for more products and reviews.
Remember to look at the Lab-things we like page for more products and reviews.
Saturday, December 15, 2012
Why we love Life Technologies Bolt gels
Lately we've been running A LOT of Western blots and our trusted Life Techologies rep, who always suggests new things to try, has been pushing the new Bolt Mini Gel Tank and the Bolt gels, so we bought one of the welcome packs (gel tank, 10 gels, running buffer, sample buffer, reducing agent and standards for $390). And a week later my tech begged for another one!
I only use Life Technologies Novex gels because despite the expense of the proprietary running buffer, they are better than the Bio-Rad ones and they were the only ones which worked well for mass spec band purification. The Novex NuPAGE gels cost the same as the Bio-Rad ones, but last longer and run very straight and crisp bands.
The Bolt tank and gels are completely redesigned. It's a double-length and narrow tank where gels are run side by side, which was strange at first, but this way you can only run a gel at a time with half the buffer. The tank also has a white background behind the gel to help you better see the loading. The Bolt gels have wells cut as wedges to make loading easier and to hold more sample than the Novex gels, AND Life Technologies lowered the price from $13 to $10 for Bolt. They run way faster, so that you can be done with a 4-12% gradient run in less than an hour. And last but not least, the bands are incredibly crisp (see the pictures comparing our samples run on the Novex vs Bolt). It kind of looks like a Christmas tree...
Note on 2/6/2013: After 3 months we found only one glitch. Running the gels at 160V as recommended makes the buffer too hot and causes some random gels to start melting deforming the front. We are lowering back to 120V as for the Novex.
Remember to look at the Lab things we like page for other reviews and cool products
I only use Life Technologies Novex gels because despite the expense of the proprietary running buffer, they are better than the Bio-Rad ones and they were the only ones which worked well for mass spec band purification. The Novex NuPAGE gels cost the same as the Bio-Rad ones, but last longer and run very straight and crisp bands.
The Bolt tank and gels are completely redesigned. It's a double-length and narrow tank where gels are run side by side, which was strange at first, but this way you can only run a gel at a time with half the buffer. The tank also has a white background behind the gel to help you better see the loading. The Bolt gels have wells cut as wedges to make loading easier and to hold more sample than the Novex gels, AND Life Technologies lowered the price from $13 to $10 for Bolt. They run way faster, so that you can be done with a 4-12% gradient run in less than an hour. And last but not least, the bands are incredibly crisp (see the pictures comparing our samples run on the Novex vs Bolt). It kind of looks like a Christmas tree...
Note on 2/6/2013: After 3 months we found only one glitch. Running the gels at 160V as recommended makes the buffer too hot and causes some random gels to start melting deforming the front. We are lowering back to 120V as for the Novex.
Remember to look at the Lab things we like page for other reviews and cool products
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