Most of these diagrams show the same thing…
The question is; have you looked closely at the differences between them and utilised all the detail from these, your lecture notes, and text books to a point where you can recreate an even better annotated diagram…?
After a fascinating debate over the ethical implications of releasing this information; the first of two papers has been published.
Very interesting article which fro what I can tell has been published in full, not censored by the National Science Advisory Board for Biosecurity (NSABB).
Do you agree with making this information publicly available…?
Particularly for 2nd years that are currently revising….
If you’re still struggling to get your head around PCR have a watch of this video. I like the way that it demonstrates the fluid nature of the reaction; it demonstrates how these biomolecules are floating around in the tube and you can start to envisage how some problems with PCR can occur if the DNA is heavily degraded or the extraction insufficiently removes potential contaminants.
No single video online is a perfect demonstration of PCR, for instance this doesn’t mention much about primer sequence/specificity and it doesn’t show the individual dNTPs before they are incorporated into the product strand. What I’m trying to get across is; don’t just rely on online videos- use these in conjunction with your lecture notes and textbooks too.
Some more of the videos from the DNALC at CSH may be worth you watching. The recombinant DNA video shows some of what I covered in my lecture on cloning into plasmids- but is missing quite a lot of info. Again, use it to put things into context and help you remember, but always be using more than one source for revision.
http://www.youtube.com/user/DNALearningCenter?feature=watch
Having just received a batch of primers for a post-grad researcher I thought I’d post a few details on handling primers- in this case received from Sigma.
Sigma provide a very detailed and useful datasheet with all their oligos which I strongly recommend reading.
Oligos are delivered dried as an almost invisible pellet- which is why they strongly recommend centrifuging the tube before opening to make sure you don’t lose your pellet! I’ve seen it done, and the aftermath of desperately trying to troubleshoot a PCR which will never work because the primer was completely lost from the stock tube.
Following centrifugation you need to elute your oligo in either ultra-pure water or TE buffer (personally I prefer water). The volume you elute to may vary depending on the desired concentration, however if you want to make life easy for yourself- there is a section on the Sigma datasheet informing you the volume to use to obtain a 100μM solution- which can then easily be diluted to working concentrations… This is usually the simplest route to take.
Note that the volumes differ for each oligo- this is because the exact amount of oligo in each tube varies slightly depending on the amount manufactured and the molecular weight of the oligo itself (being dependant on its length and base composition).
Dried primers are perfectly stable at room temperature but once eluted need to be stored in the freezer (-20°C), with aliquots of working stocks stored short term in the fridge (4°C).
There’s plenty of other useful information provided on the sheet too regarding %GC, likelihood of dimers or secondary structure, etc. all of which I suggest you take note of.
Enjoy
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I spent the morning re-optimising a PCR for a 3rd year Clinical Microbiology class and thought I’d post a quick update about the use of internal positive controls in PCR.
We all know that both positive and negative controls in all experiments are essential, the use of a positive control in PCR is your proof that in the presence of the correct target DNA, under the correct conditions the PCR will amplify, effectively telling you that both your reagents and your reaction are robust and correct.
The benefit of an internal positive control is to confirm that each individual tube is set up correctly, providing important proof that all pipetting was carried out correctly and no reagents were missed out. Internal controls are critical in plus/minus assays where you are looking for the presence or absence of a gene in a sample. Without an internal control a negative amplification would look exactly the same as a failed reaction, how would you know..?
There are a few factors to consider when trying to introduce an internal positive control. You are setting up a multiplex reaction, therefore need to make sure that none of your primers are complimentary to each other, that they all have similar enough Tm’s to anneal specifically at the same temperature, that the product lengths are different enough to be resolved on your method of electrophoresis (in this case agarose gels), yet not so different that they can’t be extended in the same length of time in the extension stage of PCR. You also need to be considering the concentration of the target sequences in the reaction. I’ve just set up a Listeria specific PCR with a universal bacterial 16S internal control for the detection of Listeria. I know that the 16S sequence can be present in multiple copies and so would preferentially amplify over the sequence coding for the fibronectin-binding protein. Therefore manipulating the primer concentrations may also be necessary to develop an optimum reaction.
A fun-filled weekend of PCR!
All the students taking the Molecular Biology module will (should) know my explanation of organic extraction inside out including me mentioning that it is sometimes necessary to vary the method based on sample type. For those I haven’t taught, or for those that slept through the lab seminars I’ll give a quick run-down of the method now, including a particularly interesting sample that cropped up in the lab recently.
This type of extraction works on most cell suspensions be it bacterial broth culture, cells suspended in some sort of lysis buffer or even in water, or from biological fluids.
Making sure you’re working in sterile, chloroform safe plasticware you mix your sample with an equal volume of TE-Saturated Phenol:Chloroform:isoamyl alcohol (25:24:1 - these numbers are the ratio of components). I tend to favour working with 750μl of sample and 750μl P:C:I in a 1.5ml eppendorf tube but you can adjust your volumes to suit your need.
In this mix you have phenol denaturing proteins forcing them precipitate, chloroform dissolving some lipids and forcing others to precipitate out, and isoamyl alcohol minimising foaming and bubbling just to make the solution easier to work with.
Give your sample a thorough mix with the solvents and centrifuge to separate the organic from the aqueous layers (see picture above). The denser organic phase of phenol:chloroform is spun to the bottom of the tube carrying with it a lot of the unwanted debris, leaving the aqueous phase containing dissolved nucleic acids on top usually with some precipitated lipids and predominantly proteins at the interface.
Careful pipetting of this top phase is essential to ensure you don’t carry over any lipids, proteins or any of the lower phase. At this stage you have the option of repeating the P:C:I (25:24:1) extraction to further purify (I know some researchers that insist on triple P:C:I extractions), alternatively you have the option of using pure chloroform, or P:C (1:1). However many variations you choose to use, the next step is generally an alcohol precipitation in order to purify and concentrate the nucleic acids.
Amongst the vast range of projects going on in the Brayford labs, a final year project this year working on peanut butter and chocolate/peanut butter sweets screening for the presence of GMOs. Using ground sample in lysis buffer (containing strong detergent) with the standard P:C:I method, the tube became completely clogged with an abundance of fats and lipids to the point the sample was unworkable; who knew peanut butter cups were so fatty?! In this case a pure chloroform extraction first was sufficient to remove the bulk of the lipids from the sample allowing the standard P:C:I extraction; a nice example of how you may need to adjust this method depending on sample type.
- As a final note: an entry on P:C:I wouldn’t be complete without me at least briefly mentioning the safety implications. Phenol and chloroform are both incredibly harsh hazardous chemicals which should be handled appropriately in a fume cupboard wearing nitrile gloves and goggles remembering to keep all chlorinated waste separate for correct disposal (tips, tubes and liquid waste)
Apologies for the lack of posts recently. This has mostly been due to me being swamped by coursework deadlines, but I do also have some developments in the pipeline for evolving this blog into something new.
As a follow up to the link I posted a couple of weeks ago; a conclusion has been made NOT to publish the details of the modified highly pathogenic H5N1 avian flu virus for now.
This has been a fascinating debate to watch unfold and leaves even some of the most experienced of scientists unsure where they stand…. I’d love to get a discussion group going on this….
To add to the obscure collection of samples which I have been asked to analyse, a couple of weeks ago I received a phonecall which went as follows; “Mike can you come and take a look at this mermaid?”……..(Me:)”Excuse me?”……”Yes this mermaid we want some DNA samples taken”. I love my job sometimes.
As it turns out this wasn’t a hoax phone call, on wandering to the offices I found this ‘mermaid’ lying on the table with a huddle of academics around it. (picture courtesy of Dave Padley).
Check out the attached news report to see some of the findings so far; the overview is that this peculiar artefact was constructed from a wire-mesh torso covered in some type of skin (as yet unconfirmed), mounted onto what appears to be the lower half of a fish and detailed with carved bone teeth. It has been suggested that it could have originated in Japan or the Far East, but identification of the fish species may help shed more light on that. So some species ID is in order. I’ve taken some samples of the ‘fish’ skin, however when this project cropped up I was in the middle of another project based on fish DNA. To absolutely eliminate the possibility of cross contamination I refused to take the mermaid sample anywhere near the lab until the previous project was completely finished and everything bleached down. Now that’s done it’s time to start work, I’ll start working on the sample this weekend and hopefully should be well underway by next week.
From the bench to Blog:
