I have inserted a gene block containing primers flanking a random sequence of 300bp into a plasmid. The plasmid was amplified and purified. We have tested it with PCR to confirm the primer sequences incorporated on the plasmid work. We have also sanger sequenced the plasmid to confirm all the correct sequences are present.
The issue we are having is that the plasmid series is not always amplifying in a dilution series from 10^9 copies/uL to 10^0 copies/uL. We have calculated the copy number in our stock using the standard copy number calculation formula based upon nanodrop values. That being said, the 10^9 amplifies very strongly and produces good banding and so does 10^8; however, once we start going down the series using 1/10 dilutions it drops off entirely sometimes at 10^7, 10^6, 10^5 etc. In that, it is not always repeatable and see absolutely no band. Sometimes the dilution series works perfectly, other times we have two bands. We need a repeatable plasmid series that works every time from 10^9 down to 10^3 or 10^2.
Has anyone else encountered such a frustrating problem and solved it? If so, what do you recommend to fix this issue?
P.S. we have regrown it, purified it and reused it to no avail. We have also linearized it and tested if this would fix the issue, no dice. If you can think of a fix, we have tried it.
Thank you so much for your help!
Hi,
there are two reasons which most probalby do synergize and give you the issuese here
1. Plasmids should be linearized to be used as template: Background is that sencondary structures might reduce the primer binding/ PCR Reaction. Therefore I would linearize the backbone. I have seen this phenomenon several times already
2. Amount of DNA is very low. I would use a dedicated buffer or a carrier nucleic acid for dilution. Apparently 10^2 copies are in the range of attogram or even lower. This DNA amounts tend to stick on surfaces. I would recommend to use something like nucleic acid dilution buffer to dilute ou plasmid. This is key to enable setup of dilution seris with low DNA amounts and plasmids.
Let me know if you need more infos on this
good luck
Sven
Jack Greenhouse
We also tested the primer issue, have used many new primers and they work fine for the target when using infected samples that we are trying to amplify which can be considered a positive control since they are known positives. So we have ruled out the primers as the causative problem.
We are using Sybr Green PowerUP from thermofisher and using a ABI 7500 Real-Time PCR Machine and the conditions are the reaction are as follows.
UDG Activation: 50C for 2 minutes
Heat activation: 95C for 2 minutes
Denaturation: 95C for 15 seconds
Annealing: 65C for 30 seconds
Extension: 72C for 1 minute
The denaturation, annealing & extension is performed for 50 cycles.
Dissociation curve is produced at the end following the instruments manufacturer recommendations.
Sven Reister
Do you recommend TE buffer for the nucleic acid dilution buffer? We have also linearized our plasmid and to no-avail. That being said, we can try linearization again and then rerun it with the dilution of TE Buffer, would non-linearized plasmid still have random amplification in which sometimes it works and sometimes it does not? Would that explain the phenomenon we are seeing?
Thank you both for your help, we really appreciate it!
Hi,
no in fact I would recommend to use something like the Nucleic Acid dilution buffer available with some QIAGEN Kits. The critical point is here to have a DNA background which prevents the absorbtion of the plasmid DNA on the walls of the tubes or the tips. As said other options might be the use of carrier RNA or non target nucleic acids (e.g. in case you are testing for a virus you might take a negative gDNA or an RNA)
Thank You Jack Greenhouse and Sven Reister we are going to try your opinion. will update you accordingly.
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