What are the conditions that need to be optimised for PCR amplification of a >1 kbp DNA fragment. I used different annealing temp. and different periods in denaturation, annealing, and extension. but I still don't get the desired size?
Nowadays, to amplify around 1 kb or a little larger then 1 kb DNA fragment should not be a problem. There are also DNA polymerase kits which can amplify much larger DNA fragment. I wish you have provided more info about your PCR set up, ex. exact size, the cycling conditions you use...etc, so we can tackle the problem.
For no results, you should pay attention to:
1. If your primers are correct
2. if PCR conditions are proper
3. If your DNA is pure enough, without PCR inhibitors
4. If your DNA template is GC rich?
5. If you need to add PCR additives to increase PCR efficiency?
6. add a positive control
7. If you add a DNA marker when you run a gel, to make sure your gel has no problem to run DNA samples (including the exist of EtBr...)
As mentioned before, it can be dependent on your specific PCR set up. Are you using a kit? Please provide any additional details.
Yuan-Yeu Yau Justin Anast
Thank you very much for your answers. They are quite useful.
the expected fragment size is1237 bp, this is to use if for cloning of VEGF whole protein. I have changed annealing temperatures to 58/60/62 and have used extended period of time (double tge original time) for denaturation, annealing and extension, yet, the maximum band size I hor is around 830 bp and it is very faint.
the kit iam using is Promega’s GoTaq® DNA Polymerase M3001
Mohamed Adel Abdelghany Abdelfattah
1. What are Tm (melting temperature) of your primers?
2. Can you attach a photo?
Considering you have specific primers:
First you should check what is Tm for your primers (online tools are available) and then try PCR reaction with annealing at 5 degree below the Tm, 30 Sec each cycle. Extension time with Taq Pol should be 1.30 -2 min per cycle.
If this doesn't work, then try gradient PCR (5 degree above and 5 degree below the Tm), you will identify the exact annealing temperature by this way.
If that also doesn't work, then try touchdown PCR, 0.5 degree decrease in temperature per cycle (20 cycles) and remaining cycles at 55 degree annealing. total cycles should not be more than 40. After running reaction product on agarose gel if you see the smear, then dilute this reaction product (1:50) and run the secondary PCR 7-8 degree below Tm, it will give you expected size amplicon.
I would also like to suggest you to check, whether your expected amplicon size is based on cDNA template or genomic DNA template as presence of intron may change the size of amplification product, please check.
Yuan-Yeu Yau
the used primer sequences is:
Fwd_Primer:
5'- GGA TCC ATG ACG GAC AGA CAG ACA GAC A -3'
Tm: 62.8 ºC
Rev_Primer:
5'- CTC GAG TCA CCG CCT CGG CTT GTC ACA -3'
Tm: 67.2 ºC
What is your template and are you certain it is ok?
Have you run a control PCR with the same kit and a control set of primers and template?
Mohamed Adel Abdelghany Abdelfattah Your Tm and Ta set up looks ok to me. Have you run a gel with your DNA sample to see if it is there and intact (not degraded)? In addition, have you checked the DNA purity? OD260/OD280 The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
The primers are not annealing with the template. Check the primers, annealing temperature, and the quality of template.
The annealing temperature for your PCR should be lower than the Tm of your primers. Make sure that you have the right primers, concentration should not be too much. Also, increase number of cycles to 30 since your expected size is more than 1 kbp.
Hope this was useful! I have troubleshooted many functional gene PCRs in my lab.
If something like you have got occurs, the reason is wrong primer design. In you case the most plausible explanation is that primers were designed to mRNA and you are trying to amplify it from DNA perhaps with introns and much longer piece. In conclusion use RTPCR with reverse transcriptase involved before amplification. By the way primers are not designed well. It not good idea to put restriction site at 5´end of the primer and their Tm difference should be lower than 4°C.
I recommend you to consider the advice of Ejaj K Pathan, Yuan Yeu Yau and Larissa Menezes given above.
I usually successfully amplify the same material with the thermal cycler setup comprised 94oC for 2 min, 35 cycles of 94oC for 30 sec, 60oC for 30 sec, and 68oC for 1 min.
If all the above advice do not work, please try to use another PCR machine (even from the same company) if you have in your lab. Because PCR machines perform differently in some cases.
I don't think this is an offtop in the thread: the Double Helix is not correct, the two strands aren't twisted, but go in parallel.
Please see my article `DNA: the Double Helix or the Ribbon Helix ?`
https://vixra.org/abs/1803.0104
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