PCR- trouble shooting - faint band in the negative control (no template) - (Apr/16/2010 )
Hello,
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
sridharagk on Apr 16 2010, 08:56 PM said:
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
band in negative control means you have a contamination problem.
When you say "prepared a fresh working stock of primers", do you mean that you make a new dilution from the stock primers in your freezer? I would order some new primers and start all over again instead of just making new work solutions from concentrate stock. Because chances are the concentrate primers are already contaminated.
Also, what's the source of your nuclease free water? I would buy it from trustworthy companies. And there's no need to sterilize the commercially available nuclease free water.
If you have access to a PCR clean hood, you may also try making your mix in the hood instead of on your bench.
Also, the source of contamination could be your pipettes or your pipette tip box. Try clean them with some DNAzap spray from Ambion as this can completely degrade any contaminating DNA or RNA.
And think about barrier pipet tips. You could also have an enzyme or enzyme buffer that is contaminated with small amounts of your template. In general, the solution involves all new reagents, water, tubes, primers. You might also avoid autoclaving tubes, since the autoclave can be a source of contamination.
ProteinWork on Apr 16 2010, 08:02 PM said:
sridharagk on Apr 16 2010, 08:56 PM said:
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
band in negative control means you have a contamination problem.
When you say "prepared a fresh working stock of primers", do you mean that you make a new dilution from the stock primers in your freezer? I would order some new primers and start all over again instead of just making new work solutions from concentrate stock. Because chances are the concentrate primers are already contaminated.
Also, what's the source of your nuclease free water? I would buy it from trustworthy companies. And there's no need to sterilize the commercially available nuclease free water.
If you have access to a PCR clean hood, you may also try making your mix in the hood instead of on your bench.
Also, the source of contamination could be your pipettes or your pipette tip box. Try clean them with some DNAzap spray from Ambion as this can completely degrade any contaminating DNA or RNA.
Thank you Very Much for your suggestions. I am using Qiagens's Nuclease free water and as you suggested. I will order new set of primers and will also perform the master mix and set up the tubes in the Hood.
I will revert back once I am done with it.
Thanks,
ProteinWork on Apr 16 2010, 08:02 PM said:
sridharagk on Apr 16 2010, 08:56 PM said:
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
band in negative control means you have a contamination problem.
When you say "prepared a fresh working stock of primers", do you mean that you make a new dilution from the stock primers in your freezer? I would order some new primers and start all over again instead of just making new work solutions from concentrate stock. Because chances are the concentrate primers are already contaminated.
Also, what's the source of your nuclease free water? I would buy it from trustworthy companies. And there's no need to sterilize the commercially available nuclease free water.
If you have access to a PCR clean hood, you may also try making your mix in the hood instead of on your bench.
Also, the source of contamination could be your pipettes or your pipette tip box. Try clean them with some DNAzap spray from Ambion as this can completely degrade any contaminating DNA or RNA.
I tried doing in the clean HOOD. But, still the same problem. I am not able to understand, what would be the problem.
ProteinWork on Apr 16 2010, 07:02 PM said:
sridharagk on Apr 16 2010, 08:56 PM said:
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
band in negative control means you have a contamination problem.
When you say "prepared a fresh working stock of primers", do you mean that you make a new dilution from the stock primers in your freezer? I would order some new primers and start all over again instead of just making new work solutions from concentrate stock. Because chances are the concentrate primers are already contaminated.
Also, what's the source of your nuclease free water? I would buy it from trustworthy companies. And there's no need to sterilize the commercially available nuclease free water.
If you have access to a PCR clean hood, you may also try making your mix in the hood instead of on your bench.
Also, the source of contamination could be your pipettes or your pipette tip box. Try clean them with some DNAzap spray from Ambion as this can completely degrade any contaminating DNA or RNA.
sridharagk on Apr 19 2010, 07:08 PM said:
Did you replace everything to eliminate the source of contamination? This includes water, primers, dNTP mix, enzyme, enzyme buffer, Mg2+ (if your buffer doesn't have Mg2+).
Also, did you clean your pipettes?
Oh, and use new PCR tubes, too.
Hi,
I see you have tried lot's of things to eliminate contamination in your negative control. I say try two more things to rule out doubt of contamination from your other wells.
1- Some times caps of PCR strip tubes gets loose and cause carry over of dna template to the negative control well in the cycler itself so -Use individual/single PCR tube(you can also cut them and make your own single tubes if you don't have any).
or
if you are using 96 well PCR plate with adhesive plate film then check/change PCR adhesive film as bad quality film sometimes cause carryover from side wells due to evaporation followed by condensation.
2-Pipette master mix first to your negative control well and then to other wells by changing pipette tip every time.
I hope this helps. Good luck.
sridharagk on Apr 19 2010, 07:08 PM said:
ProteinWork on Apr 16 2010, 07:02 PM said:
sridharagk on Apr 16 2010, 08:56 PM said:
I am doing a PCR since for almost couple years. But, since couple months, one of the gene is bugging me as I get a very faint (sometimes bright) band in the water control (no template).
primer length is between 20 - 24 bp. I have 8 different primers for the same gene (357bp).
Tm of the primers - 59 - 60 C
One primer set that I have (designed) has a Tm of 68C
Some primer sets yield a product size of less than 200 bp and some close to full length. In other words, I have a primer set that yields between 90 to 350bp. I should say, I have primers that covers almost the entire gene (357bp).
My question, is what are the best possible ways to get rid of faint band in the negative control.
I have tried the following options:
1. Prepared a fresh working stock of primers
2. Used filtered tips
3. ran the PCR in a different lab, with their pippet and tips.
4. Ordered a new taq and its buffer.
5. Tried with a different Taq and buffer
6. Prepared new working stock of dNTP's with nuclease free water
7. Sterilized nuclease free water.
Inspite of doing all there, I still a band in the negative control (no template)
I would highly appreciate, if someone could give a good suggestion in this regard.
Thanks,
Sridhara
band in negative control means you have a contamination problem.
When you say "prepared a fresh working stock of primers", do you mean that you make a new dilution from the stock primers in your freezer? I would order some new primers and start all over again instead of just making new work solutions from concentrate stock. Because chances are the concentrate primers are already contaminated.
Also, what's the source of your nuclease free water? I would buy it from trustworthy companies. And there's no need to sterilize the commercially available nuclease free water.
If you have access to a PCR clean hood, you may also try making your mix in the hood instead of on your bench.
Also, the source of contamination could be your pipettes or your pipette tip box. Try clean them with some DNAzap spray from Ambion as this can completely degrade any contaminating DNA or RNA.
hello
i'm gaby and i'm new in this field of molecular biology.actually i try to standardize the primers that i will use for the amplification of my gene.at the beginning i didn't get results and i thought that it was a pb of handling,i asked to one of my classmates to do it for me same problem,we change the bagh of gel because we used the big.after using small bagh of gel we got bands.i don't know if this idea could help you.my classmate told me too about to increase the concentration of Mgcl2 in your master mix,because she has done it and her bands appears,try to see it maybe it will be fine in your case.i'm working on it too,if i get results i will inform you.take care of you
Gaby