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-   -   inverse PCR (help me please...T_T) (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/73120-inverse-pcr-help-me-please-t_t.html)

rarhong 06-08-2010 02:49 PM

inverse PCR (help me please...T_T)
 
Does anybody doing inverse PCR here?? :unsure:
I'm not sure about the concentration to make the digested DNA circular. I read in a journal that the DNA concentration at ligation reaction must be low in order to enchance self circularization. I've tried the concentration ranging from 0,1-1 microgram/ml. But I'm afraid that small amount of DNA gone after extraction with phenol:chloroform followed by precipitation. Does anybody know any idea how to keep does DNA from missing?? My PCR always failed.
How can we know that the DNA has been circularized?? The low concentration made it difficult to see it through agarose gel.

Big big thanks for anybody who want to help.... :shy:

SebaQ 06-08-2010 09:30 PM

Re: inverse PCR (help me please...T_T)
 
Are you using Phenol:Chloroform to purify the DNA from the digestion and ligation steps?

If so, I recommend you to use a better purification procedure, like this one I shared:

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or use kits like wizard kits from promega.

Phenol:Chloroform is useful when isolating DNA from bacteria, because you have soooo many proteins to get rid of, and in much more quantities than your ligation or digestions steps. Futhermore, as bacteria has many plasmid copies and you normally process millions of bacteria, the multi-stepped phenol:chloroform isolation procedure works fine, but to keep safe low DNA concentrations, you need a protocol with less steps, for the more steps you have, the more DNA you lose between steps.

How can you know DNA has been circularized? Well, I think you can do this (you will need more concentrated DNA, so first you need to achieve that):

Take a fraction of your theorically circularized DNA and separate it in two tubes. Digest lightly one of the tubes with a restriction enzyme you do know it cuts only once in your known sequence. Let's suppose it doesn't cut anywhere else in the unknown sequence. Don't digest the other tube.

So now load in a gel both tubes: the digested one and the undigested one.

If DNA was recircularized, you will have a light "shift" in the undigested reaction compared with the digested one, because circular DNA migrates diferentially than linear DNA.

If DNA was not recircularized, in the undigested reaction you will have a band, BUT in the digested reaction you will now have 2 bands, because the linear DNA was digested in some point in your known sequence.

Hope it helps ^^!

waymouch 07-10-2013 04:03 PM

Re: inverse PCR (help me please...T_T)
 
Hello everybody
please SebaQ, I 've just a question, if DNA was not recircularized,how can I find only one band we have a lot of fragments as a result of the first digestion of genomic DNA?we should find a lot of bands isn't it?
thank you

waymouch 07-10-2013 04:10 PM

Re: inverse PCR (help me please...T_T)
 
Please concerning the inverse and nested PCR,how to choose melting temperature of primers?
for example I've these melting temperature:
iPCR: primer F : 54C
primer R : 51C
nested PCR: primer F 55C
primer R 58 C
how to choose the adequate melting temperature for each couple of primers?
thank you

SebaQ 07-26-2013 04:46 PM

Re: inverse PCR (help me please...T_T)
 
Dear Waymouch.

The number of fragments will depend uniquely on the number of restriction sites for a given enzyme within your target DNA sequence.

If there is only one restriction site in the sequence (something I suggested to do the trick I mentioned) in a circular DNA, the result will be still one band, for the circular DNA just linearized. The difference in an agarose gel between a circular and linear DNA is the migration distance. A circular DNA usually migrates differentially (supershift) than linear DNA.

On the other hand, If DNA was not recircularized and you digest it with the same enzyme you will have the two bands corresponding to each fragment you mentioned.

SebaQ 07-26-2013 04:55 PM

Re: inverse PCR (help me please...T_T)
 
Usually annealing temperatures are assumed Tm - 5 C, and it works perfectly. Since PCR usually uses primers with Tm around 50 to 60 C, effective nested PCR relies more in the effective and accurate design of the primers rather than the difference between annealing temperatures ;).

Anyway, I'd first try using 50C for your first primer pair and 55 C for the second one, though defining this annealing temperature is usually based on your results, but Tm - 5 C is a good first approach.

Hope it helps ;)!

waymouch 07-27-2013 02:24 PM

Re: inverse PCR (help me please...T_T)
 
Quote:

Originally Posted by SebaQ (Post 447733)
Dear Waymouch.

The number of fragments will depend uniquely on the number of restriction sites for a given enzyme within your target DNA sequence.

If there is only one restriction site in the sequence (something I suggested to do the trick I mentioned) in a circular DNA, the result will be still one band, for the circular DNA just linearized. The difference in an agarose gel between a circular and linear DNA is the migration distance. A circular DNA usually migrates differentially (supershift) than linear DNA.

On the other hand, If DNA was not recircularized and you digest it with the same enzyme you will have the two bands corresponding to each fragment you mentioned.

Thank you very much dear SebaQ I understood you.But let me explain what's the problem
The iPCR is done as follows:
I know that we use one restriction enzyme but it digest the whole genome so we obtain a lot of fragments with different sizes.Therefore, we'll have a ligation product with differentt sizes and so the flanking region will have different sizes.
the question now is how to know if the DNA is circulized or not knowing that we'll have many bands corresponding to the different sizes of ligation product?
thank u very much and sorry for disturbtion :)


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