Real-Time PCR Method Discussion

Real-Time PCR Method Discussion Troubleshooting forum on real-time PCR http://www.protocol-online.org/forums Wed, 08 Feb 2012 22:31:19 +0000 3600 PCR to amplify my insert not working http://www.protocol-online.org/forums/topic/24344-pcr-to-amplify-my-insert-not-working/
Im not used to doing PCR reactions so would appreciate your advice if you have any!

I am trying to change the tag on my plasmid from a Myc to a HA. In order to do this, In order to do this, I digested an empty vector containing the HA tag, and right now, I am trying to amplify my insert with the appropriate oligos.

My mentor has his own oligos for the N term and C term region and I also have a pair so I have been running both at the same time.
In order to run the PCR reaction, I am using a product called "Takara PrimeSTAR HS DNA Polymerase". The protocol they give is pretty straight forward in terms of how much to add in the master mix.

5X PrimeSTAR Buffer - 10 ul
dNTP - 4 ul (Final conc: 200 uM)
Primer 1 - 10-15 pmol (Final conc: 0.2-0.3 uM)
Primer 2 - 1015 pmol (Final conc: 0.2-0.3 uM)
Template - Less than 200 ng
Primestar HS Dna Polymerase - 0.5 ul
MilliQ - up to 50 ul

I run on a gel to confirm that it amplified there after but nothing. No bands. I ran my template, as is, and the band appears, which suggests to me that it is in tact.

Although the protocol suggests running the cycles as

98 degrees 10 sec
55 degrees 5 sec or 15 sec (depending on the Tm)
72 degrees 1 min/kb

for 30 cycles

But my mentor suggested running

98 degrees 10 sec
55 degrees 15 sec
72 degrees 5 min

for 20 cycles
and use 100 ng for template

He says that 30 cycles is too long and that it may lead to more background noise appearing?

If anyone can shed some light I would greatly appreciate it!

Thank you]]> Wed, 08 Feb 2012 22:31:19 +0000 http://www.protocol-online.org/forums/topic/24344-pcr-to-amplify-my-insert-not-working/ HRM analysis, equipment requirements http://www.protocol-online.org/forums/topic/24340-hrm-analysis-equipment-requirements/ http://www.bioer.com.cn/productcentere/productcentere?id=001001 do you think it will work for HRM analysis? Thanks in advance!!!

]]> Wed, 08 Feb 2012 15:46:59 +0000 http://www.protocol-online.org/forums/topic/24340-hrm-analysis-equipment-requirements/ AQ vs RQ http://www.protocol-online.org/forums/topic/24335-aq-vs-rq/ Do I have to create a standard curve first by dilute my samples?]]> Wed, 08 Feb 2012 12:37:31 +0000 http://www.protocol-online.org/forums/topic/24335-aq-vs-rq/ isolation of an already frozen tissue sample http://www.protocol-online.org/forums/topic/24331-isolation-of-an-already-frozen-tissue-sample/
I have a set of samples from pituitary adenomas that were put in -80 fridge (without snap freezing) right after the surgery less than a week ago without any preserving solution like RNA later or anything else. Now I want to isolate my RNA with preserving maximum quality.
What to do? I have RNA later solution and TRizol in my lab?

Should I add Trizol to the frozen tissue and immediately procede with the islolation to achieve the best results or do you have any other suggestions?
Does the fact that it was done without snap freezing will considerably affect my RNA quality?

Cheers]]> Wed, 08 Feb 2012 10:07:16 +0000 http://www.protocol-online.org/forums/topic/24331-isolation-of-an-already-frozen-tissue-sample/ <![CDATA[Problem with PCR amplification- high 3' stability]]> http://www.protocol-online.org/forums/topic/24330-problem-with-pcr-amplification-high-3-stability/ Wed, 08 Feb 2012 09:18:38 +0000 http://www.protocol-online.org/forums/topic/24330-problem-with-pcr-amplification-high-3-stability/ Home made real-time PCR problem http://www.protocol-online.org/forums/topic/24317-home-made-real-time-pcr-problem/
Many thanks.

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]]> Tue, 07 Feb 2012 15:14:00 +0000 http://www.protocol-online.org/forums/topic/24317-home-made-real-time-pcr-problem/ Pcr primers http://www.protocol-online.org/forums/topic/24289-pcr-primers/ I have to do a multiplex pcr wherein I have to add a total of 6 primer pairs to the same reaction tube.
I need to know whether I can add all primers togather and make cocktail and store them so that whenever needed i can take out that much quantity and add to the reaction mix
as otherwise this step will consume lot of time and there are chances of mistakes

Let me know what are the factors i shd consider before making a cocktail

Thanks !!!]]> Sat, 04 Feb 2012 12:54:00 +0000 http://www.protocol-online.org/forums/topic/24289-pcr-primers/ PCR failure from yeast genomic DNA http://www.protocol-online.org/forums/topic/24280-pcr-failure-from-yeast-genomic-dna/
I got a problem here... Plz help... Posted Image

I tried to delete a gene in baker's yeast by homologous recombination and need to confirm the gene replacement by PCR. I transformed yeast with the deletion cassette and a couple of candidates showed up and I picked up 6 to test for deletion.
Our group always isolate genomic DNA and do PCR to test. Normally I grow cell in rich medium YPD to isolate the genomic DNA and it worked for me almost every time.
But this time there is a plasmid I need to maintain so I grew candidate cells in selective minimal medium. I used 5ml overnight minimal medium culture for genomic DNA isolation (phenol/chloroform and glass beads vortex method), everything looked fine - I got some white DNA pellets and washed once with 70% ethanol and then dissolved in dd water.
Then I did PCR on these obtained 6 genomic DNAs along with a positive control (WT genomic DNA isolated from YPD culture). Weird thing is, the positive control showed a nice band at right size but no any band showed up for all my 6 candidate genomic DNA!!

This is the second time I observe this problem. Last time I had the same problem but after growing cells in YPD I got successful results. But this time, as I mentioned, I need selective minimal medium to maintain a plasmid (with essential gene on it).

I was careful to make sure no phenol phase was transferred, so it should not be phenol problem. I should have isolated enough genomic DNA as I could see a good amount of DNA white pellet after ethanol precipitation.

Anybody had similar experience? Probably some Taq inhibitors were carried over (somehow not a problem for YPD culture)? If so, what is the best way to remove them?

Thanks in advance.]]> Fri, 03 Feb 2012 19:13:00 +0000 http://www.protocol-online.org/forums/topic/24280-pcr-failure-from-yeast-genomic-dna/ Problems with a PCR-No bands and I am starting to question my sanity http://www.protocol-online.org/forums/topic/24260-problems-with-a-pcr-no-bands-and-i-am-starting-to-question-my-sanity/
I am having problems with this PCR, it has worked for someone else in the lab before, so this person just gave me the protocol, concentrations, I did it just like they did and it didn't work. I need to amplify the multiple cloning site of a plasmid in order to be able to sequence my insert.

I changed all the reagents, reconstituted the primers, dNTPs, MgCl2, water, buffer, got positive controls from someone else (purified plasmids)I got a new taq no so long ago. So, I did a PCR totally unrelated to this one and it worked. Yesterday, I repeated this PCR, using betaine and doing a gradient PCR and it still didn't work. I am using the following concentrations per one reaction:

*MilliQ water -25.5 ul
*Betine- 20 ul
* PCR buffer -5ul
* MgCl2 3mM- 3ul
* Primer 1 and 2 0.4 mM- 2ul each
*Polymerase (1.250 U)- 0.5ul
*DNA- 2ul (the concentration should be around 30-70nM)

Final volume: 50 ul

Thanks!!!!]]> Thu, 02 Feb 2012 19:40:30 +0000 http://www.protocol-online.org/forums/topic/24260-problems-with-a-pcr-no-bands-and-i-am-starting-to-question-my-sanity/ relative qPCR analysis when gene is not expressed at baseline http://www.protocol-online.org/forums/topic/24249-relative-qpcr-analysis-when-gene-is-not-expressed-at-baseline/
qPCR is used routinely in our lab, however I'm only just starting to seriously use it and although I've worked out most of the basics from the internet, there's a few specifics with analysing my experiments that I can't seem to work out (I've given up asking my lab to explain).

The experiments involve taking lung samples over a timecourse in mice after treatment. Some experiments have just one treatment (virus), others have two (allergen and/or virus), giving either two groups (control, virus) or four for the more complex (control, virus, allergen, allergen and virus). There are usually 4 mice per group.

I take one lung lobe and store it in RNAlater, then process it using QIAgen kits. Finally I do the RT reaction on this RNA. I Nanodrop the RNA to check concentration and that it doesn't vary too much, but I'm not currently varying what I put in the RT reaction (it's not varying too much).

What I'm wondering is this: To analyse the data I either do absolute quantification off a standard curve (which is what I'm doing for the viral load in the lung), or relative quantification via either ddCt (if the assays are equally efficient) or the standard curve method (if they're not). For relative quantification we use 18S (we'll skip over whether this is best), and to get fold change I need to use a calibrator sample, which in these experiments the control group for each time point would be ideal. However I'm looking at cytokine mRNA expression, and it's undetectable in my controls, which is not unexpected. This leads to division by zero getting ddCt, and a headache. I've tried purer cell populations with no luck in getting a baseline expression.

So, how do I analyse this data? What I think might work is:
A - normalisation of RNA into RT reaction across whole experiment and read the Ct values against a standard curve (which I have), allowing comparison along the whole timecourse and between groups (though encouraging comparison between experiments and other groups which aren't neccesarily comparable if different amounts of RNA go in) - essentially copies of gene per RNA conc.
B - report just normalised dCt, i.e. Ct(target)-Ct(18S), which I'm guessing is OK to compare along the timecourse but I'm not sure and the numbers are a bit difficult to understand at a glance as they're arbitrary
C - use a calibrator from a high-expressing mouse to get fold changes, but this seems a bit irrelevent as their treatment would be something different

I'm not sure if any of the above make sense. Either way I attempt to purify and run the whole experiment in one go, but this isn't always possible.

My second query is how would you use a calibrator sample anyway, as the 4 baseline mice aren't paired with treatment mice? Would you just average their dCt and subtract from each mouse dCt in the treatment groups so you still got statistics?

Any suggestions gratefully received! Apologies for the long post.]]> Wed, 01 Feb 2012 19:39:35 +0000 http://www.protocol-online.org/forums/topic/24249-relative-qpcr-analysis-when-gene-is-not-expressed-at-baseline/ RT-Q PCR primer design question http://www.protocol-online.org/forums/topic/24229-rt-q-pcr-primer-design-question/ So i am going to be doin Q-PCR. I am going to be using oligoDT for conversion of the mRNA to cDNA.
I am using probes and not dye for the q-PCR reaction.I am using Roche primer design assay to design assays (forward and reverse primers and probes for each gene)

Given the fact that oligoDT may not reach the 5' end of mRNA (as it only moves ~1KB), should i be designing primers/probes to be at the left or right end of the output sequence??

i was told by someone in my lab to design them as close to the right hand side as possible (presumably the 3' end), but as i put the cDNA sequence into the assay design program should it not be to the left hand side (5' end)??

Any help would be appreciated as i am getting confused.
:-s]]> Tue, 31 Jan 2012 14:19:09 +0000 http://www.protocol-online.org/forums/topic/24229-rt-q-pcr-primer-design-question/ Problems with inhibition? http://www.protocol-online.org/forums/topic/24189-problems-with-inhibition/ Thanks for the help!
Alex.

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]]> Sat, 28 Jan 2012 23:59:11 +0000 http://www.protocol-online.org/forums/topic/24189-problems-with-inhibition/ high MW dimers http://www.protocol-online.org/forums/topic/24180-high-mw-dimers/
I know maybe it could be another site the primers could've annealed to and amplified, but since it is a dimer and not a single high molecular weight band, I don't think it is another annealing site for sure. Could it be single stranded DNA? If it is, why does it happen that the PCR generates single stranded DNA ? Thanks]]> Fri, 27 Jan 2012 21:12:31 +0000 http://www.protocol-online.org/forums/topic/24180-high-mw-dimers/ Strange RT-PCR amplification plots http://www.protocol-online.org/forums/topic/24162-strange-rt-pcr-amplification-plots/
I'm relatively new, but so far I have found this site very useful! However, I have a problem I couldn't find the answer to on the forums!

I have been getting some strange amplification plots for my RT-PCR recently. I use Taqman mastermix and tailor-made probes, which have worked in the past. But, having tried a new probe I have been getting strange results (see attached) - a hump-shaped curve initially, then a gap, then the reaction proper starts later on, but for most samples it doesn't reach a plateau before the cycles end. I add a "gene of interest" probe and a "housekeeping" probe into the same reaction, and the housekeeping one works fine, it's just the gene of interest that is a problem. I am using the same cDNA and the same reaction volumes as I have successfully used before.

Any suggestions? I have already tried increasing the cycle number from 40 to 50, adding twice as much cDNA and adding 10-fold less cDNA. Should I increase the cycles further, add more probe, or add more cDNA?

Thanks!

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]]> Thu, 26 Jan 2012 14:13:48 +0000 http://www.protocol-online.org/forums/topic/24162-strange-rt-pcr-amplification-plots/ Clarifications on cDNA meaning in NCBI + Drosophila Melanogaster quantitative-re http://www.protocol-online.org/forums/topic/24149-clarifications-on-cdna-meaning-in-ncbi-drosophila-melanogaster-quantitative-reverse-transcription-pcr-using-uas-gal4-system-and-primer-design/ Posted Image

I am trying to design a primer for quantitative-reverse transcription PCR for the first time and am quite lost in the process of obtaining a 'cDNA', which derives my first and probably most important question. Below I will list out the questions I have to ask here. Thank you in advance for providing me any help with any of the questions.

1. What does cDNA refer to in the databases? Does it refer to (i) the sequence reflecting that of the mRNA (ii) the sequence complementary to the mRNA, which is the DNA sequence coding for the mRNA?

2. My work utilizes Drosophila as a model for neurodegenerative diseases and I have used a UAS-Gal4 system of overexpression for a certain gene. As such, in the context of PCR, I should be doing a quantitative-reverse transcription-PCR to measure for transcript levels and thereafter comparing it with the various controls such as wild-type Drosophila. Am I right about this?

3. Could a kind soul provide me a guide/process as to how he/she goes about designing a primer? (Or any books or references that can assist me in this?)

Thank you so much for any help rendered, I really appreciate it. Posted Image

]]> Wed, 25 Jan 2012 10:09:15 +0000 http://www.protocol-online.org/forums/topic/24149-clarifications-on-cdna-meaning-in-ncbi-drosophila-melanogaster-quantitative-reverse-transcription-pcr-using-uas-gal4-system-and-primer-design/ What products will I get from this pcr? http://www.protocol-online.org/forums/topic/24135-what-products-will-i-get-from-this-pcr/
50b Adaptor sequence - 1000b Sequence 1 - 100b overlap
and
50b Adaptor sequence - 100b overlap - 1500b Sequence 2

Thanks]]> Tue, 24 Jan 2012 13:52:31 +0000 http://www.protocol-online.org/forums/topic/24135-what-products-will-i-get-from-this-pcr/ PCR buffer without Tris http://www.protocol-online.org/forums/topic/24122-pcr-buffer-without-tris/
I am trying to do a PCR.I need to conserve the pH changes so I want to make a buffer without Tris ,so that H-ion is not buffered...I have had papers regarding this..but as they have mentioned its not working.I have tried many times ,its failure always.I make buffer with ph 8.

Any sugggestions?

Thanks
Priyanka]]> Mon, 23 Jan 2012 19:45:10 +0000 http://www.protocol-online.org/forums/topic/24122-pcr-buffer-without-tris/ PCR Water http://www.protocol-online.org/forums/topic/24121-pcr-water/
So my lab does a lot of PCR to create knockout constructs and gene tags, and over the past couple years we have had intermittent problems with contamination. Many of these problems we have narrowed down to be due to the water we use in the reactions, and consequently have tried a variety of different options, including DEPC-treated water and Type 1 Ultra-pure water from a Milipore Mili-Q filter. I was wondering if anyone else has had similar troubles with a given type/source of nuclease-free water in the past and what you have used with success.

Thanks!]]> Mon, 23 Jan 2012 16:39:37 +0000 http://www.protocol-online.org/forums/topic/24121-pcr-water/ multiplex rtqPCR and custom primer/probe design http://www.protocol-online.org/forums/topic/24116-multiplex-rtqpcr-and-custom-primerprobe-design/ I've been asked to design rtqPCRs for four cytokine assays in koalas (limited sequence available). I was wondering what the general feeling on multiplexing qPCRs is these days - particularly up to four sets + house keeping gene?

Secondly, does anyone have any experience with custom made primer/probe sets? We are on a limited time frame and were wondering if getting these designed will save us time.

Cheers
Jo]]> Mon, 23 Jan 2012 00:30:24 +0000 http://www.protocol-online.org/forums/topic/24116-multiplex-rtqpcr-and-custom-primerprobe-design/ Which Brand of real-time PCR machine http://www.protocol-online.org/forums/topic/24099-which-brand-of-real-time-pcr-machine/ My Department (India) want to purchase a new RT PCR, options are

Illumina Eco real Time 4 emission filters
ABI Step One 3 filters
Roche Light Cycler 480 6 filters

48 wells will serve the purpose,
I do not know much about these systems.
are extra filters (4/6) of much significance?
Is it that Roche system require chemicals of Roche also, than other company?

Thanks for suggestions..]]> Fri, 20 Jan 2012 18:42:29 +0000 http://www.protocol-online.org/forums/topic/24099-which-brand-of-real-time-pcr-machine/