Troubleshooting forum on real-time PCR http://www.protocol-online.org/forums/index.php Thu, 19 Nov 2009 19:31:33 -0800 3600 http://www.protocol-online.org/forums/index.php?showtopic=11676
I'm running RT-PCR for bacteria sample. My house keeping gene is 16s rDNA and I use stationary phase cultures. I have a no reverse transcritase control along with my cDNA product. So the problem is that I would have a wide difference (about 15 cycles) between the no RT control and cDNA when using 16s primers but almost no difference when i used the primers for genes of my interest. I understand that my RNA sample is probably not that clean since I do get amplification of the no RT control sample. But what would be the reason for what I see in the gene of my interest? Thanks!

]]> Thu, 19 Nov 2009 09:49:01 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11676 http://www.protocol-online.org/forums/index.php?showtopic=11674 I'd like to do my own sybr green mix with Taq gold polymerase and Sybr green 1 (purchased 10 000x in DMSO). I used to run my qPCR on CFX96, did someone do it before? Do you have any advices?
Thank you]]> Thu, 19 Nov 2009 09:21:53 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11674 http://www.protocol-online.org/forums/index.php?showtopic=11647
i'm doing qPCR and know that you have to take some normalization genes in each run.
I wondered which genes, are usually used.
Do somebody has some experience with this? And which genes, did you use?

greetz]]> Wed, 18 Nov 2009 08:33:50 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11647 http://www.protocol-online.org/forums/index.php?showtopic=11619 Good Afternoon,

We are trying to calculate PCR efficiencies for several genes that we are working with. We are using SYBR Green with a Stratagene qPCR machine. We made 6 serial dilutions of cDNA. All of our calculated efficiencies were around 80% with R2 values around 0.997. I know the acceptable efficiency is around 90-110%, but will this be a problem if we are comparing genes with similar efficiencies using the delta delta Ct method? Does anyone have any ideas on where to start to increase the efficiency? Thanks for your help.

Jon

]]> Tue, 17 Nov 2009 13:24:14 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11619 http://www.protocol-online.org/forums/index.php?showtopic=11612 Tue, 17 Nov 2009 10:20:21 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11612 http://www.protocol-online.org/forums/index.php?showtopic=11609
I'm new for qPCR and was wondering what can be the optimal target length in qPCR.
One month before I was using primers for target length of 250 and now I'm using the primers for the target length of 60. Can bad target length be problem to determine melting temperature? And can it cause any other problems?

Thanks in advance]]> Tue, 17 Nov 2009 09:19:09 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11609 http://www.protocol-online.org/forums/index.php?showtopic=11605
Iīve a problem with my real time PCR Rotor Gene 6000.

When I use Qiagen SYBR Green Master Mix, the machine works and it read the fluorescense; but when I use Fermentas SYBR Green Master Mix, this machine can not read the fluorescense. It seems to be lower than Qiagen Master Mix...

Iīve tried to change the gain (manually and automatically) for these master mix too, but still doesnīt work with Fermentas SYBR Green.

Iīve changed the master mix three times, but still doesnīt work.

I donīt know what it is... Why did Qiagen SYBR Green work and Fermentas didnīt?

Thank you for your help.


Dessy]]> Tue, 17 Nov 2009 06:22:46 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11605 http://www.protocol-online.org/forums/index.php?showtopic=11604
I have been running qPCR to test the expression level of 4 genes of interests.Now I am stuck because the efficiency of PCR is low (60~85%).Sometimes I got back very nice efficiency more than 90% which I considered can be used for analysis but it is not reproducible between batches of runs...The thing is these four genes are in one gene family so there is no much room for me to redesign optimal primers. (like GC content between 40~60%,Tm 55~60 oC etc) I have read some threads saying when using cDNA to build the standard curve it works well but when using plasmid the efficiency declines.Is it due to the inhibitor in the plasimd? But I have been using very much diluted plasmid DNA (starting from 50pg and 10-fold dilution *7) so I would not consider this point...I am using Tm of 57.4 oC based on gradient PCR.

I am using Biorad IQ5 and the mastermix they provide for SYBR.Because the primer dimer issue (in one gene of four) I add one step into the program of collecting fluorescent signal at 75 oC (15s) rather than at 72 oC (30s) for extension.Will the competition between target amplicon and primer dimer affact the efficiency?

To summarize:
1.PCR efficiency is low.
2.Did I do it right for the primer dimer issue?
3.What can I do for both issues considering I do not have much choice for my primers?

Thanks in advance for any reply to help!!!]]> Tue, 17 Nov 2009 04:22:15 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11604 http://www.protocol-online.org/forums/index.php?showtopic=11586
Used 4ul of supernatant to do PCR and I got a majority of the colonies picked to generate a band the approximate size of my insert.

When i culture the bacteria and then perform a mini-prep and double digestion to release the insert, there appears to be no plasmid at all when the gel is run. Even more, i check the concentration and all of the potential positive clones have a concentration.

How can I get a possible amplification of my insert if I don't have plasmid present? (How did the DH5 grow in the first place???)

Is the concentration just too low but the insert is there?
Is there some kind of contamination?
Did my unligated insert manage to transfect and when the DH5 lysed, was still able to serve as a template for PCR?

Any help is appreciated!]]> Mon, 16 Nov 2009 14:55:43 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11586 http://www.protocol-online.org/forums/index.php?showtopic=11580
I have been having difficulties with PCR inhibition due to plant compounds and have attempted using BSA to no avail. A colleague of mine mentioned he had used many brands of BSA before and some had worked and some didn't. Are there different types of BSA? If not why would they differ so much by brand? What type of BSA would be best for preventing plant compound inhibition?


Thanks,]]> Mon, 16 Nov 2009 12:13:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11580 http://www.protocol-online.org/forums/index.php?showtopic=11576
Right - Im doing qRTPCR in a BioRad miniopticon using the BioRad iScript sybr green master mix.

I have been getting amplification in the NTC. At first i thought that it was likely to be primer dimer as i had seen it previously on Gels so i redesigned the primers and now don't see any primer dimer either on gels or in the melt curve (So no primer dimers)

I see a single band of about the same intensity as i do on the positive control at exactly the same size. Also they have identical melt curves. (so its a specific amplification)

So the components of the reaction are the master mix, water and primers. I have changed all three and still the amplification. I did however see a drop from a ct of about 25 to 30-32ish. Meaning that some/all of those components must have been contaminated. (so have now take both my class two hood and pipettes to pieces and cleaned with ethanol/an antimicrobial spray and RNAaway spay. I have introduced new filter tips, new tubes etc. I have even changed the tape used to seal the also new plates that the reactions run in. STILL I GET THE SAME BAND.

The only thing i have possibly got left (and it seems pretty remote) is that when i opened my stocks of primers i introduced the contamination into the stocks either via the pipettes or from the hood.

Can anyone think of any other possibility.

Thanks in advance for the ideas

]]> Mon, 16 Nov 2009 07:47:12 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11576 http://www.protocol-online.org/forums/index.php?showtopic=11557 I recently changed the thermocycler i was using to run my pcr's on from one that holds .65 microlitre tubes to one that holds .2 microlitre tubes and now i've lost one of my products. I'm doing everything the same as before i've only changed the size of the tube being used and the thermocycler.
Any help on why i'm losing one of my producst would be helpful.
Cheers!]]> Sun, 15 Nov 2009 16:43:40 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11557 http://www.protocol-online.org/forums/index.php?showtopic=11554 I checked if the two suggested similar primer pairs but found none.
Maybe you can share which software are you more confident of using. Thanks!]]> Sun, 15 Nov 2009 06:05:54 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11554 http://www.protocol-online.org/forums/index.php?showtopic=11539
I have a, maybe stupid, question.
When working with real time PCR you use a molecular beacon to detect how many pcr products have been produced yet, but I was wondering: when working with a molecular beacon like a scorpion loop, when this binds to the template, is this template then "blocked" by this beacon so that it can not be used by the polymerase enzyme?

When using a taqman probe there is no problem since the taq polymerase cuts off the beacon, but what with other beacons like a scorpion beacon?
Would this beacon interupt the pcr and thus reducing the overalproduct that can be made?
Or are those beacons some how removed during the annealing after the binded to the template?

cheers]]> Sat, 14 Nov 2009 07:18:34 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11539 http://www.protocol-online.org/forums/index.php?showtopic=11533
My experiments are as follows, I have 7 different GOI and 1 HKG that I want to investigate for each of my 12 samples that are treated in different ways (+ diluted samples for standard curve, NTC etc). I want to have at least duplettes of each sample which makes it impossible to get all of the genes on one plate. So how should I do my plate setup?

Can I take one gene on each plate and be able to normalize them all against the HKG which has been run on a separate plate?

If this is ok, how about the threshold for the different runs, should they all be the same or should they be the optimal for each of the different genes?

I would be grateful for quick response since I don't really know what to do with my experiments.

]]> Fri, 13 Nov 2009 13:37:35 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11533 http://www.protocol-online.org/forums/index.php?showtopic=11525
I have also had better succes with getting Pfx to amplify genes from the extracts, however I am unable to get it to amplify my gene of interest even with control DNA.

I am looking for advice for how I may get Pfu to work on my evironmental extracts or getting Pfx to work at all. I'm really tired of messing around with this and I want my research to move forward! Help!!

~peter]]> Fri, 13 Nov 2009 11:57:20 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11525 http://www.protocol-online.org/forums/index.php?showtopic=11521
I have a little question.:
if you design expression-primers using primer-blast or another primerdesign software, is it necessary that the primers are separated by at least one intron on the corresponding genomic DNA? In primer-blast you can click on this function, but then you find less primers...

greetz,

susan]]> Fri, 13 Nov 2009 08:00:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11521 http://www.protocol-online.org/forums/index.php?showtopic=11500
It has been 2 weeks now, and I'm growing more desperate. I have the same size band in my two negative controls (NTC and noRT) and my sample, which I want to show that this gene is not present. I used a positive control, that has about 1.5 times the amount of amplicon in the exact same position on the gel. Really weird. I have decontaminated everything. The product is over 100bp, so I don't think its a primer dimer effect, and it occurs within 25 cycles. The only thing I can think of is that the TE buffer I used to dilute the primers is contaminated. Any other ideas?

Thanks, C]]> Thu, 12 Nov 2009 15:33:00 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11500 http://www.protocol-online.org/forums/index.php?showtopic=11495
Can someone please explain how I can have a Tm value for mynegative control, but not have a Ct value for the same sample?

thankyou so much.]]> Thu, 12 Nov 2009 11:18:17 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11495 http://www.protocol-online.org/forums/index.php?showtopic=11473
I am trying to use molecular methods to study bat diets, by amplifying insect DNA from bat feces. I am trying to set up a multiplex rxn to screen for consumption of three particular insects. I have designed species-specific primers for these three insects (a beetle, the primers produce a 495-bp product; a moth, the primers produce a 320-bp product; and a katydid, the primers produce a 250-bp product).

My primer pairs all work fine on their own (although the primers for the katydid are suboptimal and want to dimerize a bit; at this point, I haven't found a way around it while still keeping the specificity to the target). All the primers have Tm's between 55 and 60 degrees, GC content 40-60%. And, as I mentioned, each primer pair on its own works well to amplify DNA from target species and not from various other insects that I have screened.

Anyway, I would like to combine these into a multiplex rxn. I got a 100 rxn Multiplex kit from Qiagen, which uses their HotStart Taq in a master mix, and an optional mystery product called "Q solution" which does seem to reduce the production of non-specific products in my testing.

I started out by testing this by combining 50 uL from extractions of each of my three target species to make a positive control mix, and started testing out the multiplex with an equimolar mix of my 6 primers. I ran a few rxns with varying amounts of this template, at 57 C as suggested by Qiagen when one or more of the primers has a Tm of less than 60. (No gradient thermal cycler in my lab, boo hoo.) Template amount didn't make much difference, but what was weird was that I got a beetle band at 495 and a katydid band 250, but no moth band at 350.

I figured maybe I used the wrong moth extraction (i.e., accidentally grabbed an extraction from another moth I had extracted while testing specificity of my moth primer set) so I made sure I got the "right" moth, and added another 50ul of this to my master extraction positive control. I set up another couple of reactions with this template, one straight up, and one with a little extra of the moth primer (this is the one that wants to dimerize much more than the others). Spaced and ran them at 60C instead of 57C.

So now, when I ran out the product on the gel (from both reactions), the 320-bp moth product is there. The 495-bp beetle product is there. But now the 250-bp katydid product is missing... and it was there before!!!

Any suggestions on which next steps to take in optimizing this reaction? There are so many possible variables to adjust (template amount, primer concentration, annealing temp and time, yadda yadda) that I'm not sure where to begin. Or, since there are only three species I'm screening, should I just shrug my shoulders and run the reactions separately for each primer pair? Any suggestions appreciated, I'm a first-timer. I've uploaded the Qiagen multiplex handbook, which I have been following thus far.]]> Wed, 11 Nov 2009 12:39:20 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11473