Do you have a targeting vector that you could use as a positive control template for your PCR? It would be useful to have this to rule out degradation of your oligos. What annealing temp are you using? What is the elongation time? How many amplification cycles? How GC-rich is your target sequence? More details on your PCR protocol are needed to help troubleshoot.

You can try to decrease the Mg2Cl concentration. In this way you should be able to amplify bigger fragments.

Do you have a targeting vector that you could use as a positive control template for your PCR? It would be useful to have this to rule out degradation of your oligos. What annealing temp are you using? What is the elongation time? How many amplification cycles? How GC-rich is your target sequence? More details on your PCR protocol are needed to help troubleshoot.

Unfortunately I do not have the targeting vector. The annealing temperature is 65c and I use 35 cycles. I am checking the GC content.

The elongation time is 5 minutes with Roche expand long template polymerase and 2 minutes with Bioline ImmunMix.

For difficult to amplify sequences, I would suggest at least 2 minutes per kb. For your case, I would try a 7 minute extension time. 65C is probably too high, however, you should try to screen multiple annealing temp on a thermocycler that will let you set up a gradient of annealing temps. I would probably try 55 to 69C in 2C increments.

Finding a positive control is critical. Perhaps the company that developed the mice could provide one (even if it is just product from their positive PCR reaction).

To genotype the sequence, do you really need the whole 3.3kb? Would amplifying a smaller product be possibly easier and give you the same information?

Yes, I am wondering the same thing as Stephen. Why can't you use a smaller amplicon?

You can try OVERLAP EXTENSION PCR or Stitch the smaller fragments (with unique restriction site) after amplification !!

http://www.biotechniques.com/BiotechniquesJournal/2013/March/Gene-Splicing-by-Overlap-Extension-Tailor-Made-Genes-Using-the-Polymerase-Chain-Reaction/biotechniques-341027.html

http://www.ncbi.nlm.nih.gov/pubmed/22065455

Two thoughts

1) Scrap the assay designed by the company and design your own (i.e. pick new primers, design and optimize and assay yourself)

2) Has this mouse and its transgene already been well characterized by others? If not consider that the reason why the genotyping assay has never worked might be because the mouse and its transgene may not have been produced exactly as advertised or that there has been some breeding issue.

Good luck!

Hi,

First of all I like to ask what is the exact problem. Are you getting some non specific bands or you are getting nothing. In any case I would suggest several steps (some you may have already done).

1) Echoing Daniel, I will suggest you to check the appropriate Tm by doing Tm gradient (with different Tm)

2) You can also do touch up PCR

3) Decrease MgCl2 concentration if you dont get any product int he present scenario

4) You can also try to use smaller amplicon size

5) If all these do not work for you I can suggest you one recipe which worked for me. I used a mixture of Betaine, DTT, DMSO and BSA, I got it from a an article but I forgot the name and the authors. But the name of the complex is called preCS solution. Well, the composition is

5M Betaine 80 microlit

1M DTT 1 microlit

DMSO 10 microlit

10mg/ml BSA 1 microlit

MQ 8 microlit

Total Vol 100 microliter

use 4-10 microliter per 50 microliter reaction. I standardize the amount of the preCS as well.

Hope this will be helpful for you Good luck.

Most routine PCR genotyping protocols amplify fragments under 500bp. It would be near impossible to amplify a 3.3.Kb target from genomic DNA unless you isolate high quality genomic DNA. The easiest way to do this is to use a commercial kit for this purpose such as Clontech NucleoSpin tissue kit. You can verify that you have intact genomic DNA by running out a few microliters of your genomic DNA sample on a 1% agarose gel. You should see a single sharp band of high molecular weight with no smear in the low range. In addition you would likely require an excellent polymerase with buffers adapted for high GC content to successfully amplify complex genomic regions, especially if the primers bind near a gene promoter region, as is often the case with transgenic mice. I prefer to use Clontech/Takara LA Taq with GC Buffer I or II, a product specially designed for long range PCR from difficult templates. This approach is commonly used to amplify PCR products of several kilobases in length for PCR screening across the long homology arm region in modified ES cells (for making mutant mice by gene targeting). I recently did this for amplifying a target of ~6Kb with excellent results.

This info should help you to be able to amplify your target region of 3.3 Kb from your transgenic mice, however, I agree with John Fingert's comment that for the long term you will be much better off to always redesign the PCR genotyping protocol to amplify more reasonable size targets (ideally 200-500bp) and with primers that cover a region of the transgene that is not highly complex or GC rich. Even if you do not have the full information from the company you can do this if you simply know a partial sequence of the unique transgene.

I also concur with Markus Jager that Herculase II is incredibly robust for amplifying difficult or GC rich targets and should work in your case assuming the genomic DNA is of high quality and not degraded. I use this enzyme when others fail. However, I have noted instances where Herculase II performed better than LA Taq and vice versa in these challenging applications...FYI.

I have tried several different polymerases during last years and the best in my opinion is LongAmp Polymerase from NEB. It's also much cheaper than other polymerases and can amplify up to 30 kb. It's very good at amplifying difficult products.

We also generated 400 knock-out fusion PCR products at one go with 99% success using this polymerase (will be published soon). Check my paper below for more info about fusion PCR, if your standard PCR won't work (which was mentioned by Nishant Sharma).

https://www.researchgate.net/publication/236197011_The_cdr1B_efflux_transporter_is_associated_with_non-cyp51a-mediated_itraconazole_resistance_in_Aspergillus_fumigatus

Article The cdr1B efflux transporter is associated with Non-cyp51a-m...