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What does it mean if negative control during PCR also shows bands...
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Judas Matete
What does it mean if negative control during PCR also shows bands...
Hi everyone. happy good day for all of you. My suggestion to use the mixture without the primers and DNA template, if the band still appear; it is meaning that their is contamination ( sometimes contamination coming from your procedure not only from the reagent: especially if the bands appear sometimes or rarely. Best of luck for all of us
Hi everyone. happy good day for all of you. My suggestion to use the mixture without the primers and DNA template, if the band still appear; it is meaning that their is contamination ( sometimes contamination coming from your procedure not only from the reagent: especially if the bands appear sometimes or rarely. Best of luck for all of us
Hi everyone. I agree with Omar. At times contamination could result in the course of your procedure, i.e. allowing the tips of your pipette to touch the PCR tube during pipetting
Hi everyone. I agree with Omar. At times contamination could result in the course of your procedure, i.e. allowing the tips of your pipette to touch the PCR tube during pipetting
It means, that the pcr reaction components are contaminated. Make a fresh stock of primers, use a sterile UPW, maybe take a new pcr mix solution, and autoclaved plastic tubes. Good luck!
It means, that the pcr reaction components are contaminated. Make a fresh stock of primers, use a sterile UPW, maybe take a new pcr mix solution, and autoclaved plastic tubes. Good luck!
There is also a possibility of forming primer dimer. As there is no DNA template, primers may tend to form dimer. But most commonly contamination may occur.
There is also a possibility of forming primer dimer. As there is no DNA template, primers may tend to form dimer. But most commonly contamination may occur.
Anyone who is SURE that his/her negative control did not have contamination (Ex, prepared the negative control with newly ordered primers, PCR mix and water BEFORE dealing with any positive samples or DNA templates) , but got the correct band (better still, if you sequence-confirmed the ID), you may want to ponder whether the ghostly DNA EMF teleportation of information is real.
Anyone who is SURE that his/her negative control did not have contamination (Ex, prepared the negative control with newly ordered primers, PCR mix and water BEFORE dealing with any positive samples or DNA templates) , but got the correct band (better still, if you sequence-confirmed the ID), you may want to ponder whether the ghostly DNA EMF teleportation of information is real.
Yes it is true. The pcr materials may have been contaminated. The result from this should be discarded and a new pcr conducted under set guidelines to prevent contamination
Yes it is true. The pcr materials may have been contaminated. The result from this should be discarded and a new pcr conducted under set guidelines to prevent contamination
Hello, I agree with most of the answers, but I´m facing a similar problem. I have a band (with the expected size) on negative control and, I doubt that is contamination, because in the same set of samples I have no bands in some of them, so how it would be contamination of water or reagents? I'm using the iProof HF (bio-rad) and primers NS1/NS4 and AML1/AML2 for Glomeromycota.
Hello, I agree with most of the answers, but I´m facing a similar problem. I have a band (with the expected size) on negative control and, I doubt that is contamination, because in the same set of samples I have no bands in some of them, so how it would be contamination of water or reagents? I'm using the iProof HF (bio-rad) and primers NS1/NS4 and AML1/AML2 for Glomeromycota.
I agree. Negative control with a band different from primer dimers (very low pb) is not acceptable. How to Solve it when you can't get rid of it? You can try changing primers, designing the new ones in a way that at least one of them lays outside the former PCR product (whose presence is the main suspect here). Sometimes is the only way of stop amplifying the PCR product that is contaminating your sistem... hiding somewhere, pipets, water, etc... You can also try making a sybr-green based quantitative PCR assay, to study if the band has the same melting point, and to some extent, quantify the problem you are facing (in terms of Ct of the blank well in the real time experiment) Ajay Wagh
I agree. Negative control with a band different from primer dimers (very low pb) is not acceptable. How to Solve it when you can't get rid of it? You can try changing primers, designing the new ones in a way that at least one of them lays outside the former PCR product (whose presence is the main suspect here). Sometimes is the only way of stop amplifying the PCR product that is contaminating your sistem... hiding somewhere, pipets, water, etc... You can also try making a sybr-green based quantitative PCR assay, to study if the band has the same melting point, and to some extent, quantify the problem you are facing (in terms of Ct of the blank well in the real time experiment) Ajay Wagh
Hi Ajay, Whenever you see bands for your negative control, it generally means there was some form of contamination as Tomas has suggested. However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimer. But, if the band is the same or similar size as the positive control, it probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipetter or tips as Taras suggested.
The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipetter well since filter tips are more expensive.
Are the reagents and pipetting equipment shared by others? This also could be a source of contamination.
Hi Ajay, Whenever you see bands for your negative control, it generally means there was some form of contamination as Tomas has suggested. However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimer. But, if the band is the same or similar size as the positive control, it probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipetter or tips as Taras suggested.
The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipetter well since filter tips are more expensive.
Are the reagents and pipetting equipment shared by others? This also could be a source of contamination.
Hi everyone. happy good day for all of you.
My suggestion to use the mixture without the primers and DNA template, if the band still appear; it is meaning that their is contamination ( sometimes contamination coming from your procedure not only from the reagent: especially if the bands appear sometimes or rarely.
Best of luck for all of us
Hi everyone. happy good day for all of you.
My suggestion to use the mixture without the primers and DNA template, if the band still appear; it is meaning that their is contamination ( sometimes contamination coming from your procedure not only from the reagent: especially if the bands appear sometimes or rarely.
Best of luck for all of us
More
1
Hi everyone. I agree with Omar. At times contamination could result in the course of your procedure, i.e. allowing the tips of your pipette to touch the PCR tube during pipetting
Hi everyone. I agree with Omar. At times contamination could result in the course of your procedure, i.e. allowing the tips of your pipette to touch the PCR tube during pipetting
More
1
It means, that the pcr reaction components are contaminated. Make a fresh stock of primers, use a sterile UPW, maybe take a new pcr mix solution, and autoclaved plastic tubes. Good luck!
It means, that the pcr reaction components are contaminated. Make a fresh stock of primers, use a sterile UPW, maybe take a new pcr mix solution, and autoclaved plastic tubes. Good luck!
More
1
Negative band in the PCR simply means there is contamination of the materials used for the PCR. The PCR therefore should be repeated.
Negative band in the PCR simply means there is contamination of the materials used for the PCR. The PCR therefore should be repeated.
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VOTE
There is also a possibility of forming primer dimer. As there is no DNA template, primers may tend to form dimer. But most commonly contamination may occur.
There is also a possibility of forming primer dimer. As there is no DNA template, primers may tend to form dimer. But most commonly contamination may occur.
More
VOTE
It means that the PCR mix for the control was contaminated during the mix preparation.
It means that the PCR mix for the control was contaminated during the mix preparation.
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VOTE
Anyone who is SURE that his/her negative control did not have contamination (Ex, prepared the negative control with newly ordered primers, PCR mix and water BEFORE dealing with any positive samples or DNA templates) , but got the correct band (better still, if you sequence-confirmed the ID), you may want to ponder whether the ghostly DNA EMF teleportation of information is real.
https://www.researchgate.net/post/Has_anyone_tried_to_replicate_Luc_Montagniers_DNA_EMF_teleportation_PCR
Anyone who is SURE that his/her negative control did not have contamination (Ex, prepared the negative control with newly ordered primers, PCR mix and water BEFORE dealing with any positive samples or DNA templates) , but got the correct band (better still, if you sequence-confirmed the ID), you may want to ponder whether the ghostly DNA EMF teleportation of information is real.
https://www.researchgate.net/post/Has_anyone_tried_to_replicate_Luc_Montagniers_DNA_EMF_teleportation_PCR
More
VOTE
Yes it is true. The pcr materials may have been contaminated. The result from this should be discarded and a new pcr conducted under set guidelines to prevent contamination
Yes it is true. The pcr materials may have been contaminated. The result from this should be discarded and a new pcr conducted under set guidelines to prevent contamination
More
VOTE
Thats an indication of contamination of PCR materials. The results are not acceptable.
Thats an indication of contamination of PCR materials. The results are not acceptable.
More
VOTE
Hello,
I agree with most of the answers, but I´m facing a similar problem. I have a band (with the expected size) on negative control and, I doubt that is contamination, because in the same set of samples I have no bands in some of them, so how it would be contamination of water or reagents? I'm using the iProof HF (bio-rad) and primers NS1/NS4 and AML1/AML2 for Glomeromycota.
Hello,
I agree with most of the answers, but I´m facing a similar problem. I have a band (with the expected size) on negative control and, I doubt that is contamination, because in the same set of samples I have no bands in some of them, so how it would be contamination of water or reagents? I'm using the iProof HF (bio-rad) and primers NS1/NS4 and AML1/AML2 for Glomeromycota.
More
VOTE
I agree. Negative control with a band different from primer dimers (very low pb) is not acceptable.
How to Solve it when you can't get rid of it?
You can try changing primers, designing the new ones in a way that at least one of them lays outside the former PCR product (whose presence is the main suspect here). Sometimes is the only way of stop amplifying the PCR product that is contaminating your sistem... hiding somewhere, pipets, water, etc...
You can also try making a sybr-green based quantitative PCR assay, to study if the band has the same melting point, and to some extent, quantify the problem you are facing (in terms of Ct of the blank well in the real time experiment)
Ajay Wagh
I agree. Negative control with a band different from primer dimers (very low pb) is not acceptable.
How to Solve it when you can't get rid of it?
You can try changing primers, designing the new ones in a way that at least one of them lays outside the former PCR product (whose presence is the main suspect here). Sometimes is the only way of stop amplifying the PCR product that is contaminating your sistem... hiding somewhere, pipets, water, etc...
You can also try making a sybr-green based quantitative PCR assay, to study if the band has the same melting point, and to some extent, quantify the problem you are facing (in terms of Ct of the blank well in the real time experiment)
Ajay Wagh
More
VOTE
Hi Ajay,
Whenever you see bands for your negative control, it generally means there was some form of contamination as Tomas has suggested. However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimer. But, if the band is the same or similar size as the positive control, it probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipetter or tips as Taras suggested.
The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipetter well since filter tips are more expensive.
Are the reagents and pipetting equipment shared by others? This also could be a source of contamination.
Good luck
Hi Ajay,
Whenever you see bands for your negative control, it generally means there was some form of contamination as Tomas has suggested. However, it does depend on the size of the bands. If the bands for the negative control show products much smaller than the samples or positive control, it could possibly be primer dimer. But, if the band is the same or similar size as the positive control, it probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipetter or tips as Taras suggested.
The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipetter well since filter tips are more expensive.
Are the reagents and pipetting equipment shared by others? This also could be a source of contamination.
Good luck
More
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