Internal Positive controls...
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!
Quick look on google says up to about 15 copies of 16S operon. i.e. 4 PCR cycles worth. So if you make your 16S product relatively short then it can't steal all the nucleotides and should avoid interference with the Listeria result.
Enjoy your weekend.
It’s been a week today since I have found out I am pregnant (I’m 5 weeks at the moment YAY!).
At the moment I’m still trying to grasp on what I and cannot eat. I know the basics, no raw fish, cured meats, soft’s cheeses etc...it seems quite simple at first but there are so many other things I found out that come into it...leftovers, pre prepared salads, eggs fully cooked...so many things!!!
It’s my first pregnancy so it all seems quite overwhelming at the moment. Going from being able to eat whatever I want to no having quite a few restrictions. I just wanted to know how overly cautious I should be about this, especially with the leftovers part. This listeria business is concerning but I know it’s can be easy to go a bit over board and get really anal about what I shouldn’t eat. I know many women out there who have been preggers and eat what they like (besides the definite no-no’s )and it all turns out fine. I know I won’t be one of those ladies..especially since it’s my first pregnancy. There is definitely a degree of caution there.
So i do understand the definite "CANNOT EAT" things. But there does seems to be a few grey areas if you like...wel for me anyway...
Now..my problem is I have the sweetest Indian temp girl working with me at the moment...her brother in law owns a restaurant and she brings in wonderful curries and naan breads to share with me every day for lunch. Now the first week it was all fine. But then at the end of that week I found out I was pregnant.
If not..I’m not sure how to refuse it without being rude.....I have done so a couple of time this week, but it was so hard as she was so insistent...even though I told her I have already had my lunch. “oh please eat...I brought it especially for you..I know you will love it” I was so willing and excited when she brought in the food last week...and then all of a sudden this week I’m refusing it....I feel kind of bad.
All the food she brings are all made the night before she tells me....I dot eat quite a lot of Indian as I love it...and it tastes really fresh. So being leftovers, as long as I’ve nuked it on high...is it still ok to eat?
Or I was thinking to perhaps mention my “condition” to her so then at least she knows why. Although we haven’t told anyone yet, it being so early and all..so not really sure I want to do that. But I may have to...
As the previous poster said the curries etc are fine, just make sure you reheat it well.
The only thing I would be concerned about it reheating rice. My Ob told me that reheated rice was a big no no. Freshly cooked rice is fine, not reheated, as rice contains bacteria that can outlive the heating process. Mind you, I do it all the time now I'm not pregnant, but was cautious about it at the time.
Regarding all the other things you mentioned, its highly unlikely anything on the don't eat list will actually make you sick (cured meats etc) but I think it is sensible to avoid if you can.
I learned that Soft cheeses still have a high chance of listeria contamination and should be avoided (according to www.foodstandards.gov.au) even if they are made from pasteurized or heat treated milk. During the ripening process (when the outer coating is formed) listeria can contaminate the product and even when its wrapped/pre packaged for purchase they can still harbour the bacteria. Constant refrigeration also offers no protection against listeria as only high level heat can kill it.
I was shattered as I love Camembert cheese :-(
i had bought some Cassava Cake yesterday...i knew it wasn't as fresh as fresh could be as it wasn't as soft as usual, but have had it in the past with no problem.
I left it at my desk overnight (which i have also done in the past with no problem) only to find today that when i went to eat it it was a tiny but slimy upon me taking a bite...stupidly i took another bite as it didnt taste bad..but i knew something was not right so decided to throw it out.
Not sure if you can get Lysteria from this kind of stuff, but hopefully nothing bad happens!!!
Last edited by missmizchelle; 14-06-2012 at 15:43.
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