Monday, August 22, 2011
The End
Well I official finished my NSERC last Friday and I am now cramming for the MCATs lol. I hope you guys enjoyed what little information I posted. If you haven't already you should check out the ppt I posted, it nicely summarizes everything I have done. If anyone is thinking of applying for NSERC I would say you should definitely do it, I really enjoyed the environment and I met a bunch of cool people! I digress its been fun and if I do a NSERC next year I'll try and run the same thing however since I wont have MCAT (hopefully) I will be able to post more regularly.
Monday, August 8, 2011
Summary of research so far
So I haven't posted in a while because I've been working hard however I made this presentation for my lab meeting. It basically describes what I have been doing over the last 3 months. I hope you enjoy it.
https://www.sugarsync.com/pf/D6585058_8989944_11838
https://www.sugarsync.com/pf/D6585058_8989944_11838
Wednesday, June 22, 2011
Update on work
So far....
I have been hard at working harvesting cut plasmid and insert using various methods. I combined these in a ligation reaction and transformed some E. coli with my mixture. Unfortunately the colonies I screened did not contain my insert, meaning the vector closed without the insert. This is weird because I cut my plasmid with EcoRI and BamHI which have different recognition sites which ideally means they shouldn't get ligated back together, however we do not live in a perfect work so I suspect my plasmid wasn't completely cut with both enzymes. This would cause two things, first it would mean that its nearly impossible for my insert to be ligated into the vector and second that my vector can be ligated back together. My problem is that my restriction cut sights are so close together (2 bp) that I cannot tell by gel electrophoresis whether both enzymes have truly cut the plasmid and I have read that cut sites this close can cause problems because the RE (restriction endonucleases) cannot digest both. So to combat this I have decided to use different RE that have 20 bp inbetween them and see how that works. This means I need to redigest both my vector and insert and do gel extractions... which I dread... So I'll be hard at work the next week lol. Sorry about the lack of posts, I didn't realize how busy I would be, however I will try to post more!!
Keep commenting on my posts as it motivates me to post more!!
I have been hard at working harvesting cut plasmid and insert using various methods. I combined these in a ligation reaction and transformed some E. coli with my mixture. Unfortunately the colonies I screened did not contain my insert, meaning the vector closed without the insert. This is weird because I cut my plasmid with EcoRI and BamHI which have different recognition sites which ideally means they shouldn't get ligated back together, however we do not live in a perfect work so I suspect my plasmid wasn't completely cut with both enzymes. This would cause two things, first it would mean that its nearly impossible for my insert to be ligated into the vector and second that my vector can be ligated back together. My problem is that my restriction cut sights are so close together (2 bp) that I cannot tell by gel electrophoresis whether both enzymes have truly cut the plasmid and I have read that cut sites this close can cause problems because the RE (restriction endonucleases) cannot digest both. So to combat this I have decided to use different RE that have 20 bp inbetween them and see how that works. This means I need to redigest both my vector and insert and do gel extractions... which I dread... So I'll be hard at work the next week lol. Sorry about the lack of posts, I didn't realize how busy I would be, however I will try to post more!!
Keep commenting on my posts as it motivates me to post more!!
Protocols
I have been a little busy studying for the MCAT but I have been thinking about how to post some protocols online. I cannot make a new page so I need to post this on my main page. As these posts will probably be long and boring to anyone not interested in doing the protocol I have back-dated them so they appear at the bottom of my blog. If you use the left hand navigation tool you can click on protocols as they will all be labelled and appear here.
Saturday, June 11, 2011
Getting my NSERC position
So I first heard about NSERC though some upper year friends. I tried applying first year but I missed the dead line D'oh! So this year I decided to apply early, I emailed a bunch of profs with a brief summary of myself and my interests. Only 3 out of 15 replied to my emails, I chose the one that had the most appealing research and I asked to meet with her. It was a brief interview and by the end she asked if I wanted to work in her lab. To be honest NSERC depends completely on marks, not EC's or even the interview. You basically just find a prof willing to take you and then you and your prof write a proposal and submit it to the research department at your university. Spots are awarded based purely on your grade average. The cut off is usually around 87-89 according to my prof.
If you have any other questions feel free to write me a comment down below !
If you have any other questions feel free to write me a comment down below !
Thursday, June 9, 2011
Lab notebook
A teacher of mine once elegantly described what a lab notebook "should do." It should allow one to pick it up an repeat the experiments done by the notebook's writer and obtain similar, if not the exact same, result. I must say some people have either never heard this explanation or choose to completely ignore it.
Case and point, about half of my last week has been spent trying to get a good yield of plasmid from a agarose gel using a QIAGEN gel extraction kit. I have been following the kit protocol but have only been getting yields of around 12% whereas they quote 70-90% recovery. So after trying the extraction a couple times I asked my PI if there was anything not in the kit's manual that would increase my yield, she gave me another lab member's notebook (no longer working in the lab) to try and find what they had done to get good yields. Upon skimming through and finding a similar gel extraction I observe that their yields are ~50%, so I decide to look for the protocol they used. After reading the entire notebook I realize that the only "protocol" they have is "gel extraction was done, here are the nanodrop readings". I am utterly amazed how little detail this person put in their lab notebook. So I now have to try a number of different tricks I found on the internet to try and boost my yield while wasting reagents, wasting my time, when clearly this person optimized the protocol! Their ignorance in not writing it down has meant all the time THEY spent optimizing it is effectively wasted! What is more annoying is that this isn't the only example, there are numerous places where more detailed is need yet to accurate replicate their experiments!
This is PSA!! Please keep detailed lab notebooks! It not only benefits you, but the entire scientific community!!
Case and point, about half of my last week has been spent trying to get a good yield of plasmid from a agarose gel using a QIAGEN gel extraction kit. I have been following the kit protocol but have only been getting yields of around 12% whereas they quote 70-90% recovery. So after trying the extraction a couple times I asked my PI if there was anything not in the kit's manual that would increase my yield, she gave me another lab member's notebook (no longer working in the lab) to try and find what they had done to get good yields. Upon skimming through and finding a similar gel extraction I observe that their yields are ~50%, so I decide to look for the protocol they used. After reading the entire notebook I realize that the only "protocol" they have is "gel extraction was done, here are the nanodrop readings". I am utterly amazed how little detail this person put in their lab notebook. So I now have to try a number of different tricks I found on the internet to try and boost my yield while wasting reagents, wasting my time, when clearly this person optimized the protocol! Their ignorance in not writing it down has meant all the time THEY spent optimizing it is effectively wasted! What is more annoying is that this isn't the only example, there are numerous places where more detailed is need yet to accurate replicate their experiments!
This is PSA!! Please keep detailed lab notebooks! It not only benefits you, but the entire scientific community!!
Wednesday, June 8, 2011
Welcome!!
Hello to all!!!
This is the first post on my new blog "Summer of a NSERC student." I have decided to start this blog with the intent to give other students some idea of what a undergrad student would do day-to-day working under a NSERC USRA grant. My hope is to update weekly, if not more often, with reports of what life is like working 9-5 in a lab. I don't want to give any specifics about the position or myself but sufficed to say I just finished second year and I'm in a Clinical Biochemistry stream at my university. My hopes are to apply to write the MCAT this summer and apply to Medical school this upcoming cycle.!!
I would love to hear any of your comments, suggestions for improvement or questions!
PS. here is information about NSERC USRA http://www.nserc-crsng.gc.ca/Students-Etudiants/UG-PC/USRA-BRPC_eng.asp .
- it is basically a government program I applied to in order to get funding to work in a lab for the summer months (16 weeks)
This is the first post on my new blog "Summer of a NSERC student." I have decided to start this blog with the intent to give other students some idea of what a undergrad student would do day-to-day working under a NSERC USRA grant. My hope is to update weekly, if not more often, with reports of what life is like working 9-5 in a lab. I don't want to give any specifics about the position or myself but sufficed to say I just finished second year and I'm in a Clinical Biochemistry stream at my university. My hopes are to apply to write the MCAT this summer and apply to Medical school this upcoming cycle.!!
I would love to hear any of your comments, suggestions for improvement or questions!
PS. here is information about NSERC USRA http://www.nserc-crsng.gc.ca/Students-Etudiants/UG-PC/USRA-BRPC_eng.asp .
- it is basically a government program I applied to in order to get funding to work in a lab for the summer months (16 weeks)
Summary of work so far.
So I realize I might be starting this blog a little late, since I've already worked for about 4 weeks, however I feel I can adequately summarize what I have done so far. I'm writing this post with a understanding that most people reading it will have a background in science, however if you don't know something or feel something is unclear feel free to ask!! I have decided to break it up into two sections as follows...
"Bench Work" Aspect
Week 1-2
- Spent most of my time in the lab's student office reading scientific articles relating to my labs principle interest.
Learned a few techniques such as:
- E. Coli transformation (i.e. getting an E. Coli cell to uptake a plasmid)
- plasmid mini prep (i.e. harvesting plasmid from transformed E. Coli). This involves using a "kit" which is basically just a set of prepared solutions and elution columns that make harvesting plasmid easy. The aim of this process is to obtain a "stock" of plasmid which I can subsequently use for other experiments where I will ligate "MFG" into the plasmid, then transform E. Coli with the plasmid and get them to express "MFG"
- Gel extraction (i.e. taking a band out of a agarose electrophoresis gel and retrieving the DNA contained inside). I will use this technique at various points throughout my project to purify DNA
- DNA digestion with restriction enzymes
Week 3-4
- So basically for the last 2 weeks I have been trouble-shooting the above techniques to obtain optimal yield for each process and to ensure the products I obtained are suitable for further experiments.
Non "Bench Work" Aspect
What really surprised me is the amount of time my lab spends in what I would call "professional development". We spend about 6-8 hours a week doing various activities such as "journal club" (where one student presents a critique of a predetermined scientific article and subsequently the entire lab discusses the paper), going to talks where prominent guest speakers present their current research and attending conferences devoted to one topic where various guest speakers present their work and students present posters of their current work.
"Bench Work" Aspect
Week 1-2
- Spent most of my time in the lab's student office reading scientific articles relating to my labs principle interest.
Learned a few techniques such as:
- E. Coli transformation (i.e. getting an E. Coli cell to uptake a plasmid)
- plasmid mini prep (i.e. harvesting plasmid from transformed E. Coli). This involves using a "kit" which is basically just a set of prepared solutions and elution columns that make harvesting plasmid easy. The aim of this process is to obtain a "stock" of plasmid which I can subsequently use for other experiments where I will ligate "MFG" into the plasmid, then transform E. Coli with the plasmid and get them to express "MFG"
- Gel extraction (i.e. taking a band out of a agarose electrophoresis gel and retrieving the DNA contained inside). I will use this technique at various points throughout my project to purify DNA
- DNA digestion with restriction enzymes
Week 3-4
- So basically for the last 2 weeks I have been trouble-shooting the above techniques to obtain optimal yield for each process and to ensure the products I obtained are suitable for further experiments.
Non "Bench Work" Aspect
What really surprised me is the amount of time my lab spends in what I would call "professional development". We spend about 6-8 hours a week doing various activities such as "journal club" (where one student presents a critique of a predetermined scientific article and subsequently the entire lab discusses the paper), going to talks where prominent guest speakers present their current research and attending conferences devoted to one topic where various guest speakers present their work and students present posters of their current work.
MCAT resources!
Just thought I'd share some resources I have found
http://www.wikipremed.com/ has some amazing links !
Good Self Study Guide that I am following!
http://forums.studentdoctor.net/showthread.php?t=801917
http://www.wikipremed.com/ has some amazing links !
Good Self Study Guide that I am following!
http://forums.studentdoctor.net/showthread.php?t=801917
Wednesday, June 1, 2011
Protocols
Here is the first protocol I learned (adapted from Sigma Manual)
Sigma Mini Prep protocol:
Before beginning:
- Make sure Wash solution has ethanol added
- Add RNase to Resuspension solution
Protocol:
1. Pellet 1-5ml of overnight culture
2. Resuspend in 200ul of RNase Resuspension solution
3. Add 200ul of lysis solution and let sit for up to 5 minutes, mix by inverting not centrifuge
4. Add 350ul of neutralization solution and mix by inverting the tube
5. Centrifuge at 13000rpm for 10 minutes
6. While waiting, prepare column by adding 750ul of column prep solution, centrifuge at 13000 for 30-60s and discard flow-through
7. Remove supernatant and place into column
8. Centrifuge column for 30-60s (13000rpm)
9. Discard flow through
10. Add 750ul of Wash solution and centrifuge for 30-60s at 13000rpm
11. Discard flow through, Spin again for 30-60s at 13000rpm and discard flow through
12. Apply 50ul of EB buffer directly to column filter and let stand for 5 minutes
13. Spin column for 30-60s at 13000rpm
14. Flow through contains DNA, quantify on nanodrop.
Pl Please let me know if you like this and I will post more, otherwise I will stop.
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