Touch down PCR - (Sep/11/2006 )
Hi everyone,
I have been doing this touch down PCR and I am using Tris and KCl which I made myself. I tried 2 different 10x Buffers but they never gave me any products. When I tried the Tris (pH 9) and the KCl I get the products I want, but the problem is there is a shadow under my bands when I run a gel. I want to send the PCR products for sequencing but the company says, those shadows will interfere with the sequencing. Does anyone know what this shadows are and what can I do to get rid of them? I'll really appreaciate the help, thanks.
Bongiwe
it would help to know the size, as they may be primer-dimer?
but my first guess would be degradation products. are all these lanes done with the same chromosomal prep? perhaps there was some minor shearing, leading to some smaller, truncated products
one possibility is to run the gel out as far as you can, then cut and purify your bands from the gel. some of your sequencing reactions may not give you good results if there are multiple products, though.
best would be to re-prep your DNA with very careful technique, PCR again and see if you can clean up the bands
What touchdown temps were you using? And what number of cycles?
You might want to try starting at a slightly higher initial annealing temp to see if you get rid of those bands.
Thinking about this for a second, if the shadow bands are truncations of your target sequence, one of two things will happen. If the truncation is at the sequencing primer end, you won't get any signal. If it's at the other end, the signal will dropp off as soon as you reach the end of the truncation. Conversely, if the bands are unrelated to your sequence, you probably won't get any response from them at all...
Maybe.
you may also do your pcr with DMSO 1% for start, but may range from 1 to 5%.
generally avoiding the non specificHow did you prepare your samples for electrophoresis? there may be a bad soluton that gaves you degradation.
you may also heat briefly your sample to 65° before loading it. will hels to separate secondary structures that may be present in your sample.
Can't you just send 1 sample to sequence and see what you get out of it? It's hard to tell exactly what you're seeing; so why not give it a shot? (just one reaction with one primer, shouldn't be too expensive?)
Hey all, is my thinking wrong here....
For this situation I would simply gel purify and extract the desired band. I wouldn't care about all the other products being generated as I would consider them side products. In fact the DNA bands look strong and sharp enough that I would just make more DNA fragment, carefully run a nice well loaded gel with good separation, and cut the gel just below the bright band leaving behind its lighter tail.
Rather then go through all the trouble of optimising PCR conditions.
Awww, come on, where's your sense of adventure? What about the joy of spending weeks chasing after the culprit, only to find it was because the third-year student gave you tap water rather than the sterile MilliQ you requested?
Actually, that might just work, if you can get good resolution.
Bongiwe, what gel conditions did you use for that gel image?
Rather then go through all the trouble of optimising PCR conditions.
Awww, come on, where's your sense of adventure? What about the joy of spending weeks chasing after the culprit, only to find it was because the third-year student gave you tap water rather than the sterile MilliQ you requested?
Actually, that might just work, if you can get good resolution.
Bongiwe, what gel conditions did you use for that gel image?
It died under the withering glare of my supervisor. He sits besides me, with the provobial whip in hand.
"Stroke! Stroke! Ye, galley slaves. *SNAP* Stroke! Put yer backs into it!"
You can almost hear it.
Odd isn't it, that many things become funny on the retelling.
By the way, I have taken a look at several pictures of my gels. Most have those faint bands at the lower end of the gel. I don't actually bother about them. Just gel extract and be off with it.
EDIT: Pauses... ops... rereads past post, my mistake. Got confused for a second.
"Stroke! Stroke! Ye, galley slaves. *SNAP* Stroke! Put yer backs into it!"
You can almost hear it.
Odd isn't it, that many things become funny on the retelling.
By the way, I have taken a look at several pictures of my gels. Most have those faint bands at the lower end of the gel. I don't actually bother about them. Just gel extract and be off with it.