Any chance the primers or the master mix have gone bad? I think most PCR master mixes have a much shorter shelf life than the separate components, and I know I used to have primers go bad after ~6 months when I stored them in distilled water instead of TE buffer, even at -20°

Any chance the primers or the master mix have gone bad? I think most PCR master mixes have a much shorter shelf life than the separate components, and I know I used to have primers go bad after ~6 months when I stored them in distilled water instead of TE buffer, even at -20°

Hi,

Check your buffer, ions, and polymerase. If you are diluting anything or preparing something like buffer, use deionized dH2O.

Alexandra Johnson

Thank for your reply, Alexandra. The primers are not that old, they have been ordered 3-4 months ago. Also I have tried a new Q5 MM but still have the same problem. Like the chance is controlling the PCR, sometimes I get the specific band with some other unspecific , sometimes not and I get only the unspecific ones. NB: Usually the unspecific band is brighter that's why I thought maybe the primers are not very good. I used another pair of primers but still unable to get the specific product size.

Shamsunnahar Mukta

Hi Shamsunnahar,

I use the Q5 MM as it is. The only diluted components are the template (5ng/ul), the primers (1:10) and I use NFW for the dilution.

If you are happy that the primer quality is good then the next best candidate is the template dna. As it is plasmid we can assume that the quality is good but there is still a potential problem with secondary structure interfering with the pcr. It would be good to linearise the vector with an enzyme digest but it is likely that you cannot do this but you could try running the pcr in presence of 6%DMSO to minimise secondary structure loops and twists which may interfere with primer extension. Secondary structure is very temperature dependent which might be why it only sometimes happens. 1M betaine in the reaction mix may have a similar effect

Paul Rutland

Thanks a lot Dr. Paul for your reply. I have used Gotaq master mix (taq polymerase) to see if the problem is in the conditions or in the primers and the template. With Gotaq I got very bright specific bands at a range of different Ta with the other unspecific bands were very faint. My guess is that Gotaq although has lower fidelity than Q5 it is more tolerant to the variation in conditions than Q5. But I still need to get the product using Q5 for further work afterwards. what do you suggest?

How much template DNA are you using for template Osama Kamal . Plasmids are very small so small amounts have very large copy numbers so use 1picogramme to 10nanogrammes ( hugely less than genomic dna).I did not see what volume of 5ng/ul you are using in the reaction It may be that you are using too much template . The recommended annealing temperature for q5 is 3c above the Tm of the lowest annealing primer . The proof reading ability of Q5 does slow the processivity of the enzyme quite a lot so you may want to increase the extension time to favour the production of longer amplimers

Denaturation 6-10seconds at 98c. Elongation times of 50-60seconds /kb may help.

Thanks Dr. Paul Rutland . For the Q5 reaction I use 1 ul of the (5ng/ul plasmid) for a total reaction volume of 25 ul. I use 1min:30sec as extension time at 72C for 4600 bp product. Denaturation 98 C for 10 sec, annealing 1 degree above the lower primer Tm ( I also tried higher Ta) for 30 seconds.

Osama Kamal I do appreciate that the q5 manual suggests very short extension times for simple templates and plasmids and that 1.5mins looks like it is long enough but you seem to be doing so much correctly that a longer extension time still seems worth a try with less dna in case the dna has any Mg chelators as contaminants which may be removing some Mg from the reaction. I assume that you are using thin walled pcr tubes or strips allowing the short denaturation time to be effective. Are you using heated lid or oil overlay? If oil overlay then the oil volume must be very small or the thermal inertia of the mix plus oil volume may mean that the reagents do dot reach the desired temperatures.

I am using heated lid cycler and PCR tubes strips. I can give it a try with increasing the extension time to 2 minutes let's say. Paul Rutland

Possibly a couple of dilutions at lower amounts of template Osama Kamal and maybe one with the GC enhancer to remove secondary structure issues

Thanks a lot Dr. Paul Rutland . Will give it a go and let you know the outcome. Cheers

Hi:

If you use Q5, please use the TM from the link below. the Q5 has a much higher TM compare with taq.

https://tmcalculator.neb.com/#!/main

Primers should be:

•Not containing inverted repeat sequences to avoid formation of internal structures.

•40-60% GC content preferred for better annealing.

•Tm of primers can be calculated to determine annealing T0

•Primer is an oligonucleotide sequence – will target a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids.

•PCR primers are short, single stranded DNA molecules (15-40 bp).

•As you design primers with a longer length (15 → 40 bp), the primers become more selective.

–As the size of the primer is increased, for example, a primer sequence of 35 base letters repeatedly encountering a perfect complementary section on the target DNA.

1.Universal – amplifies ALL bacterial DNA for instance.

2.Group Specific – amplify all denitrifies for instance.

3.Specific – amplify just a given sequence.

Junjie Shao Thanks. I indeed use it.