7 replies to this topic

[thedanielrecord]

Recently I've been having this weird problem with my PCR. I have a 1.5kb insert in the Topo PCR 2.1 plasmid. I digest out my insert with EcoR1 and gel purify using the Qiagen Gel Extraction Kit. I check to see if I have a product in my gel ( I do ) and then I run a digest using PST1 of the 1.5kb insert. I visualize the digested bands ( a 1 kb fragment and 0.5 kb fragment ) and they're there, clean and neat. I extract the 1 kb band, the 0.5 kb band and the area of the 1.5 kb fragment. I PCR amplify using primers that should only amplify the 1.5 kb fragment ( I'm trying to prove that I have complete digestion, which I don't think I'm getting ) but I get huge smears.

here's the weird part. I have 3 positive controls : the plasmid with the insert, the purified insert ( from earlier ) and a genomic sample that will be amplified by primers. The Plasmid and the genomic sample work just fine but the purified insert does not. The purified pcr control looks exactly the same as my digested products.

I've changed all my reagents, all my kits and done this reaction in parallel with a PhD in my lab and the results are identical. Why would an insert amplify with while in a plasmid but not amplify when it's been digested out? Mind you there are no extra EcoR1 sites in the insert and the EcoR1 site is about 50 bp away from the insert. Further more, I've gotten this reaction to work as little as 2 weeks ago. I have multiple aliquots of this DNA and I've switched them out as well with the same results. I've run it across 5 different aliquots and everyone causes this huge smear. I'm going crazy.

[eldon]

hey...i'd like to help, but for some reason, i don't get the point of your experiment...testing complete digestion of a purified insert?

your pcr doesn't sound optimal...how clean is your area where you gel purify? use less template in your pcr....is the smear above and below 1.5kbp?

hotstart? touchdown?

[thedanielrecord]

eldon, on Jun 18 2009, 09:46 AM, said:

hey...i'd like to help, but for some reason, i don't get the point of your experiment...testing complete digestion of a purified insert?

your pcr doesn't sound optimal...how clean is your area where you gel purify? use less template in your pcr....is the smear above and below 1.5kbp?

hotstart? touchdown?

Ah, so the point of the experiment is to answer whether I'm getting complete digestion of this 1.5 kb insert. One of my committee members says I am while I think we are not. He wants me to try this experiment which honestly, I'm not sure will not prove anything ( i mean, what if the reason why I'm not getting amplification is because I was unable to recover any DNA, not because I had complete digestion ).

My PCR should be optimized. normally when I've run this reaction , whether it's using genomic dna , plasmid with the insert or just the purified insert, I get one incredibly strong band that's about 1.5 kb. it's very very clean.  This smear thing is only recently and because of it, I can't prove my hypothesis because nothing is being amplified.  I've varied the amount of DNA from as small as 20 pg/25 ul reaction to 24ng/50 ul reaction and I have the same result.

The smear goes from the well all the way down, so it does not seem if the smear is the result of the 1.5 kb fragment being digested.

Again, the key thing is that the experiment worked as little as two weeks ago, it's just now giving me problems. I changed out my taq as well today ( phusion high fidelity taq ) and the problem remains.

Thanks

[eldon]

thedanielrecord, on Jun 18 2009, 12:04 PM, said:

eldon, on Jun 18 2009, 09:46 AM, said:

hey...i'd like to help, but for some reason, i don't get the point of your experiment...testing complete digestion of a purified insert?

your pcr doesn't sound optimal...how clean is your area where you gel purify? use less template in your pcr....is the smear above and below 1.5kbp?

hotstart? touchdown?

Ah, so the point of the experiment is to answer whether I'm getting complete digestion of this 1.5 kb insert. One of my committee members says I am while I think we are not. He wants me to try this experiment which honestly, I'm not sure will not prove anything ( i mean, what if the reason why I'm not getting amplification is because I was unable to recover any DNA, not because I had complete digestion ).

My PCR should be optimized. normally when I've run this reaction , whether it's using genomic dna , plasmid with the insert or just the purified insert, I get one incredibly strong band that's about 1.5 kb. it's very very clean.  This smear thing is only recently and because of it, I can't prove my hypothesis because nothing is being amplified.  I've varied the amount of DNA from as small as 20 pg/25 ul reaction to 24ng/50 ul reaction and I have the same result.

The smear goes from the well all the way down, so it does not seem if the smear is the result of the 1.5 kb fragment being digested.

Again, the key thing is that the experiment worked as little as two weeks ago, it's just now giving me problems. I changed out my taq as well today ( phusion high fidelity taq ) and the problem remains.

Thanks

get a new advisor. i still don't see the point...and it seems like you're doing unnecessary PCRs and spending unnecessary time and $$$ on something...do you need the 1 and 0.5 fragments for a real experiment? probes? why not PCR amplify the 1 and 0.5 kb fragments from the vector that houses the 1.5 kbp insert if you just need the fragments

minimize the extension time

did you sequence your insert?

how do your gel purified fragments look when you view them? do you do an overexposure...you might be surprised what you see.

you must be purifying non-specific contaminating gDNA....try spiking the digested 1.5kbp with 10 ng of the vector housing the insert and see if the smear disappears...the specificity of your insert/vector will out compete contaminating bacterial gDNA if that's the problem..

[egerecske]

hi, I have a very similar problem, the difference is that I have to produce longer products (up to 6.5 kb), and I have to make several micrograms with PCR. I have three fragments, call them A, B and C which have to be amplifed for further sequencing. With my test-template, all of them worked (genomic DNA). Later I tried it with other samples, some worked, some not. After a lot of failures, I don´t know what happened, fragment C amplified from ALL of my templates, but A and B NOT (usually 2-4 out of 8-10 is working though in A and B as well, not always the same samples for the two fragment). So what I conclude:
- cannot be the template, because it worked for fragment C (and it would be weird to think that ALL my DNA strands in my stock are broken/degraded in fragment A and B but not in C...)
- cannot be the primer, because it works in some sample in each run
- cannot be the mix (dNTP, enzyme whatever), because samples from the same mastermix behave differently.

I reduced template, for I thoutght the main reason for such a smear can be due to this. In fact, I got less and mopre pale smears, but they are still tehre, even if I use gDNA now in a much lower concentration than before, and at the beginning of this experiment those samples WORKED. I mean, now I´m only going on with samples that ever seemed to work with all (or most) of my primers. I also combined it with touchdown, still smears are in many lanes, and I have samples where I have now more than one band (they are usually lower in size).
But I really have to get some result very soon so please if you have any idea (and not cloning) I´d apprectaite it very much! It really drives me crazy now.

[egerecske]

I wouldn´t care about the smear itself, I mena I´d reduce my samlpes even more... what is too bad, that there is no product in certain lanes, while I know that there _was_ a reaction when _that_ sample worked with _that_ primer.
I think this was the reason why I increased the starting template, which caused smear...

[phage434]

I would immediately redesign your primers.  The fact that they work in one amplification does not mean they are particularly good primers.
PCR is a weird way to try to establish RE cutting -- almost certainly you will pick up full length product, because the reaction never goes to completion, and the PCR will amplify what is left.  Quantitative PCR might be worth doing, however.

[egerecske]

ok, maybe my primers are not perfect but tehre was not nly one working reaction, but several - seemed to me that I can have around 20 samples having most of the fragments I need. And now in every sample I got a smear, quite strong smear. And the bands are not there anymore. I checked the DNA quality again, most of the samples are good qualty, though some has a very low concentration. how yould I make the smears disappear...?