i have used earlier in takara mister mix for amplification to amplify target gene and gene size is 2415 bp. but now i have been using different enzyme for amplification and also same cyclic condition. i got multiple band whereas target band also very thin.what is the reason for this multiple band? What is the optimum conceentration of DNA template for PCR reaction to amplify target gene?
The 'multiple bands' can be associated with several factors. The list below might present just a few of them:
1. The enzyme, which you said 'it has been changed to other brand'. Which one are you current using? If it is a low 'processivity' enzyme and only suitable for a shorter distance amplification, then it might give you trouble.
2. Your Ta (annealing temperature), a low Ta of your primers might pick up some non-specific bands
3. Your primers may not be unique enough
4. Your gDNA may be not pure enough (with some contaminants)
5. Some PCR additives can be added to enhance the 'specificity' and 'efficiency' of PCR performance
Maybe the new enzyme has a lower synthesis rate? You can also try to enhance the annealing specifity by addition of 5% DMSO. Mayby toch-down-PCR is also a good idea to eliminate unspecific bands.
1-10ng of template in a 50uL reaction is fairly standard. More is not good. Although amplification can lower starting amounts is feasible, it is not recommended for cloning as you increase the likelihood of PCR errors with more extensive amplification.
Different enzymes work in different reaction buffers that can affect the stringency of your oligo hybridization, especially due to changes in magnesium chloride concentration. It is not uncommon to see different PCR banding patterns and specificity of amplification using different polymerases. For cloning a larger cDNA (>2kb), you had best stick with a high fidelity enzyme (Phusion, Pfx, Pfu Turbo, etc).
Hi there,
The amount of template will depend on its nature. If it's a plasmid, from 1pg to10ng in 50µL reaction is OK as Daniel said but if it's genomic DNA you will have to consider hundreds of ng(up to 250ng per 50µL) has your target will be much more diluted in gDNA than in a plasmid.
Use LongAmp Taq DNA Polymerase and set up the extension temperature on 65C for 1 minute per 1000 bp. First step is to do an annealing gradient temperature to find out the best temp for your primers that gives you a good yield with low non-specific products. Although 10ng of input DNA concentration is most commonly used, but for such size (2415bp) I would suggest to use higher concentrations of input DNA (30-50ng) in 20 µL reaction mixture.
thanks to all for giving valuable suggestion. i will fellow and then inform you. before 6 month back i have amplified and confirmed in same gene by sequencing at 60O C for 1 mint in annealing temperature and final extension is 10 mints. takara master mix was used but now i have used topo enzyme,Pfx, Pfu ....the band coming very thin and also multiple bands
Hi Mani
For long-range PCR between 10 and 30ng should suffice within a 50 microliter reaction. Be sure to do QC on your DNA template first, this may be degraded and the problem. Gradient PCR, changing magnesium chloride concentration should give you a better idea of what's going on. Adjuvant addition such as DMSO (10% usually) or formamide (1 % usually as this reduces the amount of target amplicon produced) might also allow for better amplification. Careful here though as this may affect your downstream applications (e.g. NGS etc.) If that fails you might need to redesign your primers.
Goodluck
Usually the template concentrationficient of 100-200ng/ rxn will be suf
You should probably run a minus template negative control to determine whether the incorrectly sized PCR products are coming from contamination with other DNA or primers. If the Takara kit worked well before, why switch now?
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