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These are the instructions for use the spreadsheet.
If you only have one biological replicate for now that's fine you can do the other ones later. The only thing you have to repeat every time is the no template control ("blanks").
We use an Excel spreadsheet from Biorad for our ct/expression analysis, though there are probably better/easier ways to do this. It's not the greatest (eg. your data has to be layed out perfectly before importing it) but we've been using it for years and once you get the hang of it, it's easy. You just need to input the ct values so you can take data generated at any time from any machine. Most qPCR machines should be able to output an excel sheet or txt file with the ct values (we use a Opticon Monitor from Biorad, also an old program). It basically automates your calculations, including normalizing to a housekeeping gene and calculating fold change. If you leave efficiencies at 100% you're using the ddct method, if you calculate efficiencies using a standard curve and give that data to the spreadsheet you're using the Pfaffl method (information on the two methods, at the bottom of this page).
Here's the spreadsheet:
and here's some instructions:
This spreadsheet shows on the left how I would organize my data before importing it into the spreadsheet from Biorad, and on the right where each value would end up once imported:
Everything in the first three columns in red would be copied then imported in to the Biorad spreadsheet. Note how each is labelled and how the coordinates are organized.
Otherwise nested primers sound like a good idea. Without nested primers, the primers for the second round of PCR have to bind right at the very end of the template. Maybe a bit of degradation knocks a base pair or two off the end making binding much less efficient (I'm just guessing here). I know that restriction enzymes don't bind efficiently right at the end of a DNA strand, maybe primers don't like to either (more guessing ).
I read in a forum a while ago that someone would take a needle and poke it into the gel into the PCR product band, then just stick the needle tip into the PCR mix for the second reaction. Enough DNA would be transferred just on the needle tip to reamplify it. I've never tried this so I can't comment on how well it works.
I will do that in same plate, but annealing temperature for each gene is different. Therefore I can't run them on same time.
You don't have to run all your genes at the same time. You don't even have to run them on the same day. As long as you're consistent and proficient with your PCR set up you'll get the same results whether you run them together or seperately. You should however keep your biologcal samples together ie. if you're comparing expression of a given gene in one normal vs one stress condition, run both that normal and stress condition for that gene in the same run. If you're not sure just how proficient you are, then repeat some of your PCR after a few days and see if it's consistent.
I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.
In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.
I am unable to figure it out, can you please help me.
I've had situations like this. Sometimes you'll see just one band on the gel but get multiple peaks in the melting curve. This could be because there are actually two peaks on the gel but they are too close together (running your gel longer may help) or there could be faint secondary products that you can't see on the gel, as the realtime PCR melt curve is a lot more sensitive that an agarose gel. Unless both my gel and melt curve indicate one clear product I don't trust the results and I either optimize the primers or design new ones. You could try an annealing temperature gradient and see how that affects what the melt curve and gel look like.
I set up a gridded plate and a complete PCR mix in uncapped PCR tubes (strips or plates). I use a sterilized pipette tip just held in my clean gloved hand to scrape off the colony, scrape a bit onto the gridded plate, then place in a PCR tube and let it sit there until I've collected all the colonies. An 8x8 gridded plate works well with 8-tube PCR strips. It's very fast, and you'll get enough cells on both the gridded plate for more bacteria to grow and in the PCR mix for a strong PCR reaction.
I originally would just put in in water then add a few uL of the water to the PCR mix, which worked, then to go faster I tried with a PCR master mix minus the Taq (didn't want Taq sitting in the MM for so long) and that worked, so I went ahead with the Taq pre-added and it also worked. I'm not even using hot-start Taq. If I have more than one plate to do I'll split it up so the MM + Taq doesn't sit at RT for more than a few minutes. ie. do one plate's worth of reactions in one PCR block and start it, then set up a new MM for each subsequent plate.
What are your cycling parameters? With a 284 bp amplicon you can extend for 20-30 seconds even with low quality Taq, less for better Taq. This may help limit formation of the bigger, non-specific bands.
If you use an SDS lysis buffer from the start you can perform all the lysis and sonication in the same buffer, but you may get interference or background from cytoplasmic proteins.
Here's some good information:
I hope I explained that ok, it's kinda confusing. I don't know any specific reading material I just learned bisulfite sequencing from others in my lab and the lab next to us. Feel free to ask for any clarifications.
In regards to storing cDNA, it's more hardy than you think. I've never heard of storing it at -80. I store my cDNA at -20 and freeze/thaw it many times without any degradation. The problem I describe above occurs from immediately after the cDNA is made, it's not a freeze/thaw issue. I've forgotten cDNA in the fridge for several days, and at room temperature overnight, without any problems (although I wouldn't recommend that!).