PCR Troubleshooting - (Sep/10/2007 )
Hi,
I am trying to genotype mice via PCR. More specifically, I am looking for knockouts. When I started, I was given mice and told to test for two genes, first was the WT gene, the second is the gene that was inserted to replace the WT gene.
I can find the WT gene fine and dandy.
The gene that was inserted is giving me a lot of problems.
I have made new primers and tried the buffer/dNTP/Taq protocol handed down to me, as well as another one from a labmate.
My problem is that I keep getting an amplicon in WT samples for the replacement gene - which is not found in WT animals, but in bacteria. When I get amplicons for the WT samples, my negative control (dH2O) is clean. This occurs for the old primers that were handed down to me. When I get an amplicon in the WT sample, I also get amplicons in the samples I'm testing.
However, in the new primers, I don't get any amplicons in the WT sample nor the other samples, also with a clean negative control.
I use the same WT sample for both primer sets. I've starting using a different PCR program for the new primers and will be reversing which primers go with which program the next time I run samples to test the programs.
My PI says that there must be some positive control contamination in my WT samples and that one set of primers is able to detect it but the other set is not. Is this possible?
I disagree with my PI. I know I have not contaminated my samples - at least, if they are contaminated, it is not due to dirty pipette tips or leaving the tubes open. I close everything immediately after I use it and am very careful to change tips every time I add something to the master mix.
Could I have contamination in my buffer/dNTP/Taq/MgCl2/primers and still get a clean negative control?
I am at my wits end. I don't know why one set of primers gives me an amplicon and another doesn't. I feel like I have tried everything. I clean my pipettes before I use them, I change my dH2O, I make new primer dilutions and new DNA dilutions everytime I start a new run. My PI wants me to even clean the PCR machine. How am I supposed to do that, Q-Tips dipped in 70% ethanol? I don't think the contamination is coming from the machine - the tubes are never open while inside.
I need help!
I have a question, Is you old primers that you inherited different from your new primers.
What I am trying to ask is are oligonucleotide sequence of your old primers different from the new primers?
If the sequence are different then their sensitivity can be different. Some primer pairs do primer better then others.
My suggestion is take everything that you have and throw it into the bin. Start fresh, make new primer dilutions (I hope you have been very careful with the master primer stocks), new Taq, new buffer, new MgCl2. (thus the importance of making small aliquote)
1 . Are you absolutely sure your WT samples aren't Exp samples, the transformants? Could there be a mix up? (Just checking... best to cover the basics)
2. The old primers that you have inherited, could they be contaminated? Could the main stock be contaminated?
3. Or worst yet, it may also be possible that your genomic template is actually contaminated. Did you process your Wild type and experimentals at the same time? Could there be any contect the bacterial gene, which I assume was put into a plasmid first.
What I am trying to ask is are oligonucleotide sequence of your old primers different from the new primers?
If the sequence are different then their sensitivity can be different. Some primer pairs do primer better then others.
My suggestion is take everything that you have and throw it into the bin. Start fresh, make new primer dilutions (I hope you have been very careful with the master primer stocks), new Taq, new buffer, new MgCl2. (thus the importance of making small aliquote)
1 . Are you absolutely sure your WT samples aren't Exp samples, the transformants? Could there be a mix up? (Just checking... best to cover the basics)
2. The old primers that you have inherited, could they be contaminated? Could the main stock be contaminated?
3. Or worst yet, it may also be possible that your genomic template is actually contaminated. Did you process your Wild type and experimentals at the same time? Could there be any contect the bacterial gene, which I assume was put into a plasmid first.
So my old primers are different from my new primers, they produce different amplicons that are about 20-30 bps apart.
I've tried to start fresh many times and get the same results. I am going to be trying that again soon, I'm waiting on orders to come in.
For your questions:
* Yes; we began to suspect that some of the WT samples I inherited may not have been true WT samples, so I have taken new samples from true WTs. Still get a band with the old primers and nothing with the new.
* I have ordered new batches of the old primers, but it is possible that they may be contaminated by now, I guess. But the negative control comes out clean.
* For this question, I assume you mean did I purfiy the DNA of the WT and exp. samples at the same time? If so, then no, with the samples I am using now, they were purified separately. However, I do run the samples at the same time when I run the PCR. As for the contact to the bacterial gene, I don't think the samples have come in contact. I keep my tubes closed until I need them and I don't use the same tips twice. My positive control comes from bacterial gene, but this has been made for a long time. The experimental samples, I believe, may have had the gene inserted via a plasma, but I don't know, this was done before I got here.