Hi
I want to amplify the fragment of 2500 bp, but everytime it failed. I performed touchdown PCR and I got amplicon size of 500 bp. What might be the reason that I am unable to get the desired fragment. Can I think based on 500 bp fragment that the taq polymerase failed to extend and amplify or whether one of the primer (forward or reverse) bind the template and other has a nonspecific binding resulting in 500 bp amplication? Please enlighten me....
Start with looking at how your gDNA is, if the extraction has resulted in high fragmentation, then there is no way you are going to get a product you desire,
Then followup with primer design.
Finally with using specific kits for long range PCR.
Usually for larger products you need to work on extension time of the PCR based on the processivity of ur polymerase enzyme. Looking at the booklet that came with ur enzyme will help immensely. Also an increase in the denaturation time during pcr cycle might be required.
Hope this helps,
JS
- It might be that standard taq compared with other highly processive enzymes has indeed just made a 500bp fragment, although fragments of the wrong size might also be non specific
- In the context of standard WT taq making fragments above 2kb in my experience is not as consistent and efficient as making fragment below that size: The single most effective alteration you could make is to use a highly processive polymerase like NEB phusion
https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase
- Whether in the context of phusion and but also Taq also pay attention to your PCR cycling conditions; In particular
A. Commence PCR with a single cycle of 96C for 5 minutes
B. Add 5% DMSO to your PCR: This will also help reduce secondary structure that is inevitable in larger amplicons
C. In the main body of your 3 step PCR, denature @ 96C not 94C and also denature for 20-30seconds not 10-15 seconds as prescribed in standard Taq based PCRs
D. It is inevitable that larger (>2kB) products in general will be made with less efficiency and thus in smaller amounts than say a 500bp product. Thus amplify for 35 cycles instead of the usual 25-30
Thank you Edel, Laurence, and Siddharath for your kind advice. I am indebted to Edel for suggesting such a fabulous paper.
Hi Qurshid,
I would also add that you might want to try alternative primer combinations. Especially if you are targeting a potentially higher copy locus, you may be amplifying a partial duplication of your target at another location in the genome.
Good luck!
I agree that other polymerase or another primers can help. In some cases PCR produces short amplificate and there are no problems after change of one primer producing longer amlificate. The reason may by some repetition.
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