I'm using gradient PCR with Dream Taq polymerase enzyme and mentioned PCR conditions and components in protocol.
I'm getting a high intensity non- specific band lower than the expected amplicon length at highest temperature and I got a low intensity band at same length at calculated annealing temperature whereas rest all empty.
*Taq Buffer contains 20mM of MgCl2
Now, How can I optimise the conditions or PCR components ?
You might get rid of the non specific band by running the pcr with DMSO at up to 8%. If you have some of the correct size band then you could try either re amplifying with nested primers or separating the band on agarose gel,then re amplifying the correct size amplimer diluted to about 1/100 and run different numbers of cycles from 10-20 cycles and see which ones give good amplification.
If all that you are getting is the low size amplimer then re designing your primers is probably the best try. How small is the small band? could it be primer dimer or is it an amplified product?
It's not the primer dimer..It's an uncharacterized product..I did primer blast and checked..Band was near 1.2 kb
Hi,
I suggest to do a Touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for exmple 65-70 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the calculated annealing temp of your primers. For example 55 °C (lowest temp). Run the PCR on this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique not as elegant as gradient PCR, but it can be usefull. Unf you won't know the ideal annealing temp like in grad PCR, but it will maximize the amount of the specific product.
Bests,
Tamás
You may use an alternative polymerase enzyme. DMSO is not so efficient in amplifying GC-rich regions! I recommend Tamás and Paul statements in advance. Best luck
Dear Gayathri Kks,
When you asked a question like that, you need to give people more details/information, so people can give you better suggestions. A picture is always welcome, even a failed one.
The info should include:
1. What is your DNA source? Genomic DNA? plasmid DNA?
2. Are your DNA clean? Purity checked? Contaminated with PCR inhibitors if its purity is low?
3. Your gene--GC rich or not?
4. What is your expected size of your DNA (you already mentioned it, ~1.2 kb)
5. The Tm (melting temperature) of your primers?
6. The PCR cycling parameters you set [especially, the anneal Temperature (Ta)?) and extension time?]?
7. Did you include a positive control and a negative control? How did they look? Did the positive control good? And did the negative control clean (no band at all)??
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