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PCR Trouble Shooting - new primer, wrong dilution of primers, wrong band, and many more (Jun/13/2007 )

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QUOTE (Nabin @ Jun 18 2007, 10:54 AM)
Cheamps and Swannny,

Thank you. Your suggestions are making me understand many things I had never thought about PCR. I had other programmes in the weekend so could not be concentrated on this though I tried some of them.

The primers didn't give equal bands first and I had them adjusted till I got almost equal looking bands.

The calculator is cool, Cheamps. I was relying on the data sheet of the manufacturer. The forward primer (GALFDJH) was supposed to have Tm at 85deg and reverse primers (NeoU and BGLGAL) were supposed to have them at 75.6 and 74.1 deg C. The annealing temp the paper I referred suggested using 55 deg C as annealing Temp and was wondering why so much difference. But, when I calculated with the calculator I found them to be 68.5, 60, and 55.4 deg cel. Which should I be taking as the true Tm? Why is there so much difference between the one provided by the manufacturer and the one that was calculated?

Another question (may be very basic question) - how do I find the sequence of primer without the restriction site or additional site?

I tried Swanny suggested Touch down PCR but I got it all mixed up and ended in a mess with primer-dimers. I will try fixing and running again. But, may be after a few days as I have a presentation to make today.

Thank you for all your help.

Hi Nabin,
Tm calculations vary because different companies use different equations. Personally, I always took the rough-and-ready approach and said 4 deg for each purine, and 2 deg for each pyrimidine (swanny now prepares for small firestorm of flame-mails for being lazy and not using a 'proper' equation). Remember, the PCR annealing temperature is set 5 to 10 degrees below the Tm, so a slight miscalculation of a couple of degrees shouldn't matter. You might want to talk with the company who made the primers and ask why there is a 15 to 20 degree difference between their calculated Tm and yours.

A restriction site sequence will be palindromic, and if you're lucky, it'll be something common, like EcoRI (GAATTC), HindIII (AAGCTT) or BamHI (GGATCC). Does the paper tell you what site was incorporated? Typically, you'll have a few bases at the extreme 5' end of the primer that allow correct digestion by the enzyme, then the site itself, any other sequence desired, and finally the template-specific sequence.

I can't imagine what the somewhat 'cryptic 'another' site could be, but, again, I'd imagine the paper should give you the correct details.

Happy amplifying.


Thank U again for clearing my confusions. I was busy with my data presentation regarding my real project so didnt have time to try PCR again and I did some trials again today - compared the primers, checked if the samples contain DNA or not (it did give bands for another gene), ran samples with 3 primers (forward primer 1 and reverse 2 for the wild and knock out genes), tried with primers separated in pairs, ran samples at different annealing temp, tried changing annealing time to 1 min and extension time to 1min30sec . .. .and also did protocol like swanny suggested (is it called the touch down PCR?) . . . but nothing really helps. . . and wild DNA is on and off. May be I should consider a major revision and find another primer.

I had one question in mind. The paper I consulted said 781bp for the KO gene; but I got band at around 1200bp. This band was sharp and present only in KO sample but not in the wild sample. I tried with wild, my positive sample, and another KO mouse sample that also has Neo primer. The KO band at 1200bp was seen only in the sample I considered positive while in the other two it remained negative while for wild gene it was positive for other two gene but absent in the KO gene.

So, I can distinguish between my KO mice gene and wild gene and another mouse with KO of different gene. It serves my purpose. But, the band is not at 780bp. What can be the cause? And, is it ok to depend on this band and test my mice to check which one has the KO gene I am looking for? Or, does the band size matter and I should stick strictly to the paper I referred?

Today overnight I am trying again the original protocol but only 35 cycles. Let me see what the results will be.


You can't because you don't know what you are sequencing. It could be a strain specific allele for all you know. It could also hint that some scrambling has occured.

However, if you believe the band is specific to the KO gene, that try sequencing the 1200bp band. That way you can find out what it is.


QUOTE (perneseblue @ Jun 22 2007, 04:06 AM)
You can't because you don't know what you are sequencing. It could be a strain specific allele for all you know. It could also hint that some scrambling has occured.
However, if you believe the band is specific to the KO gene, that try sequencing the 1200bp band. That way you can find out what it is.

What is 'strain specific allele' and 'scrambling'?

I don't know how sequencing is done but it's good time that I learn.


QUOTE (Nabin @ Jun 22 2007, 02:44 AM)
What is 'strain specific allele' and 'scrambling'?

Ah, sorry. It was a random example of a unlikely horror senario.
What i mean was that 1200bp fragment may be cause by something other then your KO gene. The primcers could be binding to some other region in the genome, which by chance is the same only in the knock out mice. Or it could mean that your knockout gene is not cleanly knocked out. Something has happened to the structure.

But the point , is that the 1200bp is not what your expect. (Look this up again. Are you sure you should be expecting 780bp?). As a result you can't be certain what you are sequencing.


May be I should learn more about genetics and PCR before I attempt such things. I don't do molecular biology except this PCR to check my mice babies and all I was doing was following protocol which was left to me by a senior that always worked. But, now that I have a another knockout, I looked for article about that and found primers and the whole protocol there. So, attempted!!!! But things didnt work out.

The paper says 780bp so I think 780bp. I will have to ask some one to do sequencing other wise.

Basic things I still don't know are many like. . . how to know what primers to use and how to know how big should the product be.

May be I should quit here and work on theory first.

Thank U everyone for ur help. I have wider knowledge but little knowledge is always dangerous. Swanny wished me 'Happy Amplifying' but hope everyone will also Amplify Happiness,.


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