I am going to clone gDNA fragment > 10 kb into a pCR TOPO vector. I performed the PCR using KOD PLUS Neo polymerase for 3-steps PCR reaction, I am not able to find the specific size. Several an specific bands were founded while I start by gradient PCR reactions from 54-64 degree
I am looking for an appropriate polymerase /or experiment condition to amplify gDNA for such size. OR Can anyone please share their experience.
Thanks :)
Hi Ammar,
I have been able to amplify ~9500 bp fragment from Agrobacterium gDNA using KAPA HiFi Polymerase and MgCl2 at [2.5 mM]. I used a touch down+expand (-0.5degC/cycle and +30sec/cycle) for the first 10 cycles with primer anneal at 60degC for 20sec and extension at 72degC for 10 min. The next 25 cycles were 55degC anneal for 20 sec and extension at 72degC for 15min. Hope this helps. The KAPA suite of polymerases are fantastic.
Hello Dr.Ammar,
To my knowledge, KOD-Plus-Neo can amplify targets more than 10 kb (according to the company, its up to 17.5 kb). There are several other high fidelity polymerases under different names developed by many companies. In the past, I used Tks Gflex™ DNA Polymerase from Takara for amplification of long target (more than 10kb). You can see the details for this polymerase here:
https://catalog.takara-bio.co.jp/PDFS/R060A_DS_j.pdf
Recently I am using polymerases developed by New England Biolabs (NEB). They have several options for polymerase based on your experiment. You can check the list with features here:
https://international.neb.com/tools-and-resources/selection-charts/pcr-selection-chart
Don't feel restricted to these polymerases. You can check other companies and see their list of high fidelity polymerase and their features.
However, looking at the smeared band in your gel, I don't think your problem is the polymerase. I think you need to optimize your PCR. There are many possible reasons I can think of:
- First, in my experience, smeared DNA usually caused by a high concentration of template DNA. Try to use serial dilution to see if it works.
- The second most common reason is DNA contamination/carry-over ( if the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents)
- Your template DNA is degraded
- Too many PCR cycles (Reduce the number of cycles)
- Non-optimal Mg++ concentration
- Nucleotide concentration is too high or unbalanced
- The primer annealing temperature is too low (even with gradient PCR, low annealing can cause aspecific bands, try to optimize it according to your primers s Tm)
- Mispriming caused by the secondary structure of template, snapback, or excessive homology at 3' ends of primers
- DNase activity (indicated by smears visible on gel below expected band size)
- Incorrect template to enzyme ratio (enzyme concentration too high)
For a start, I recommend decreasing the cycle number and using less template DNA. Other things, 1.Increase the denaturation time and temp. 2. Reduce extension time. 3. Increase annealing temperature 4. Decrease the enzyme and mg concentration 5. Check for contamination using negative control
One more thing, check your primers using BLAST to confirm whether they can bind to more than one site in your genomic DNA or not.
Hi, any high fidelity enzyme with a proof reading activity should be able to amplify a 10 kb region. My favourite is phusion polymerase. However, to amplify a 10 kb region needs lots of optimization. To ensure successful PCR, I would recommend for high annealing temp of around 65 C. You can also add DMSO that would help in getting the 10 kb band.
Another way of cloning would be using Gibson assembly that might be more appropriate in your expt.
Ammar, I'd suggest as first step to give the polymerase enough time to do its job and then to optimize annealing temps and other conditions.
To this end, try to amplify a known fragment of similar size with primers for which you know the annealing conditions (maybe you have a similar insert that is flanked by T7/Sp6 sites).
Once the extension time is fine, proceed with annealing conditions. If your cycler supports touch down, go for it, it is usually the quickest way to get the job done.
Joshua, Sayanta, Wolfgang thanks a million , I may try to use KAPAHiFi Polymerase or phusion polymerase.
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