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Troubleshooting: Inverse PCR


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#1 djvan

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Posted 08 December 2013 - 12:57 PM

Hello - I recently designed an Inverse PCR to amplify a segment of unknown DNA flanking an are of known sequence.  If you're unfamiliar with the process, this picture from Wikipedia sums it up pretty accurately: http://upload.wikime...Inverse_PCR.png  The main difference between my IPCR and the picture if that I did not restrict the known sequence area (Red in the picture).

 

Briefly, I did the following:

 

1) Restricted whole genomic DNA with an RE (ApoI) that cut on either side of the known sequence area.

2) Choloroform/Phenol Extracted, and Ethanol Precipitated DNA

3) Ligated DNA at varying concentrations.  The goal here is to get your restriction of interest to ligate with itself and form a plasmid.

4) Choloroform/Phenol Extracted, and Ethanol Precipitated DNA

5) PCR.  A reaction was run for each of the chosen ligation concentrations.  Controls for each concentration were also run - these simply used the originally primers that amplified the known sequence.

 

Results:

No amplification - not even in the controls.  

 

To troubleshoot I modified the above - I eliminated all Chloroform/Phenol Extraction and Ethanol Precipitation.  Now my procedure looked like this:

 

1) Restricted whole genomic DNA with an RE (ApoI) that cut on either side of the known sequence area.

2) Heated killed restriction enzyme according to the manufacturer instructions (80C, 20 minutes).

2) Ligated DNA at varying concentrations.  The goal here is to get your restriction of interest to ligate with itself and form a plasmid.

3) PCR.  A reaction was run for each of the chosen ligation concentrations.  Controls for each concentration were also run - these simply used the originally primers that amplified the known sequence.

 

Results:

No amplification with inverse primers.  All controls (using inward primers) generated amplicons.  This was true for all the concentrations used during ligation.

 

Here's my thinking:

 

1) ApoI and it's buffer and ligase and its buffer, do not inhibit PCR.

2) Heat killing ApoI may be ineffective in reducing its activity.

3) ApoI and/or it's buffer may inhibit ligase activity.

 

 

The flanking amplification should be a ~800 bp, which shouldn't be a problem for "normal" Taq.  I'm trying to avoid chloroform extraction and ethanol precipitation, because I believe I'm losing my DNA during these steps.  Does anyone know of a good protocol for chloroform extraction and ethanol precipitation when using low DNA concentrations?

 

Thanks for any help, and I'm happy to answer any questions - I'm sure I've left certain things out.

 

Jake



#2 phage434

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Posted 08 December 2013 - 05:54 PM

It sounds like your ligation is not working. You could try a test ligation with a plasmid containing an ApoI site, just to make sure you can ligate the cut fragments. Why did you choose ApoI over some more common, highly efficient enzyme?

 

Can you tell us your ligation conditions and DNA concentrations you are using? Enzyme, volumes, concentrations, everything.

 

You can likely get your Phenol chloroform extractions to work well by precipitating with glycogen, linear polyacrylamide, or Novagen Pellet Paint.



#3 djvan

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Posted 09 December 2013 - 07:40 AM

Phage, thank you for your response and interest in helping me figure this out.  Here's a bit more information:

 

I'm working on characterizing a large deletion in a model organism.  Hence, the restriction enzymes I selected were based off the wild type animal, whose sequence downstream is known.  The enzymes cut on either side of my "known" sequence in WT, and result, if succesfuly ligated, in a plasmid anywhere between 800-3000 bp.  I've selected 4 REs: ApoI, BspHI, EaeI, and NsiI.  The first time I ran the reactions (with chloroform/phenol and ethanol) I used all 4 REs - in separate tubes, of course.  I didn't get any amplification.

 

To simplify things, while I troubleshoot, I'm only using one restriction enzymes.  I have a fair amount extra of ApoI, that's why I'm troubleshooting with it.

 

My ligations reactions look like this:

 

10x T4 Buffer: 5uL

T4 Ligase: 1uL (~400 Units)

DNA: 10ng, 30ng, 50ng, 100ng, 200ng, or 500ng (0.5uL - 25uL)

H2O: up to 50uL

 

The package insert says ligations can occur at room temp for 15 minutes, or at 16 C for 12-16 hours.  I tried both methods - same results.

 

Also, the lack of amplification using my inward, control, primers, indicates that there is no DNA in my sample - pretty sure I lost it somewhere in the chloroform/phenol and ethanol.  I'll take a look into the glycogen precipitations and novagen pellet paint.  Running the DNA on a gel won't be helpful, as I do not know what fragment size to expect in the mutated model.



#4 Ramo711

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Posted 31 January 2014 - 01:25 PM

djvan, have you been able to make any progress on this issue?

I am using a similar set up with a few adjustments:

Restriction Enzyme: HindIII

5X T4 Buffer - 20uL
T4 Ligase: 1uL
DNA: 100ng, 325ng, 550ng, 775ng, 1000ng (1uL -10uL)
H2O: up to 100 uL

For the ligation reaction I leave it at 16 degrees for 16 hours. And have replaced the chloroform/phenol extraction with heat inactivation.

Thanks






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